Anti-HER3/HER4 antibodies binding to the beta-hairpin of HER3 and the beta-hairpin of HER4

ABSTRACT

The invention relates to anti-HER3/HER4 antigen binding proteins, e.g. anti-HER3/HER4 antibodies, that bind to the beta-hairpin of HER3 and the beta-hairpin of HER4, methods for selecting these antigen binding proteins, their preparation and use as medicament.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/073,137, filed Nov. 13, 2013, now U.S. Pat. No. 9,725,511, whichclaims priority to European Patent Application No. EP 12191871.8, filedNov. 8, 2012, the disclosures of both which are incorporated herein byreference in their entirety.

SEQUENCE LISTING

The present application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 2, 2017, isnamed P31325-US-1.txt and is 124,906 bytes in size.

FIELD OF THE INVENTION

The invention relates to anti-HER3/HER4 antigen binding proteins, e.g.anti-HER3/HER4 antibodies, that bind to the beta-hairpin of HER3 and thebeta-hairpin of HER4, methods for selecting these antigen bindingproteins, their preparation and use as medicament.

BACKGROUND OF THE INVENTION

The HER protein family consists of 4 members HER1, also named epidermalgrowth factor receptor (EGFR) or ErbB-1, HER2, also named ErbB-2,ErbB-3, also named HER3 and ErbB-4, also named HER4. The ErbB familyproteins are receptor tyrosine kinases and represent important mediatorsof cell growth, differentiation and survival. The HER family representreceptors proteins of different ligands like the neuregulin (NRG)family, amphiregulin, EGF and (TGF-a). Heregulin (also called HRG orneuregulin NRG-1) is e.g. a ligand for HER3 and HER4.

Human HER3 (ErbB-3, ERBB3, c-erbB-3,c-erbB3, receptor tyrosine-proteinkinase erbB-3, SEQ ID NO: 3) encodes a member of the epidermal growthfactor receptor (EGFR) family of receptor tyrosine kinases which alsoincludes HER1 (also known as EGFR), HER2, and HER4 (Kraus, M. H. et al,PNAS 86 (1989) 9193-9197; Plowman, G. D. et al, PNAS 87 (1990)4905-4909; Kraus, M. H. et al, PNAS 90 (1993) 2900-2904). Like theprototypical epidermal growth factor receptor, the transmembranereceptor HER3 consists of an extracellular ligand-binding domain (ECD),a dimerization domain within the ECD, a transmembrane domain, anintracellular protein tyrosine kinase domain (TKD) and a C-terminalphosphorylation domain. This membrane-bound protein has a Heregulin(HRG) binding domain within the extracellular domain but not an activekinase domain. It therefore can bind this ligand but not convey thesignal into the cell through protein phosphorylation. However, it doesform heterodimers with other HER family members which do have kinaseactivity. Heterodimerization leads to the activation of thereceptor-mediated signaling pathway and transphosphorylation of itsintracellular domain. Dimer formation between HER family members expandsthe signaling potential of HER3 and is a means not only for signaldiversification but also signal amplification. For example the HER2/HER3heterodimer induces one of the most important mitogenic signals via thePI3K and AKT pathway among HER family members (Sliwkowski M. X., et al,J. Biol. Chem. 269 (1994) 14661-14665; Alimandi M, et al, Oncogene. 10(1995) 1813-1821; Hellyer, N. J., J. Biol. Chem. 276 (2001) 42153-4261;Singer, E., J. Biol. Chem. 276 (2001) 44266-44274; Schaefer, K. L.,Neoplasia 8 (2006) 613-622) For an overview of HER3 and its variousinteractions within the HER receptor family and the NGR ligands familysee e.g. G Sithanandam et al Cancer Gene Therapy (2008) 15, 413-448.

Amplification of this gene and/or overexpression of its protein havebeen reported in numerous cancers, including prostate, bladder, andbreast tumors. Alternate transcriptional splice variants encodingdifferent isoforms have been characterized. One isoform lacks theintermembrane region and is secreted outside the cell. This form acts tomodulate the activity of the membrane-bound form. Additional splicevariants have also been reported, but they have not been thoroughlycharacterized.

Interestingly in its equilibrium state, the HER3 receptor exists in its“closed confirmation”, which does mean, the heterodimerizationHER3beta-hairpin motive is tethered via non-covalent interactions to theHER3ECD domain IV (see FIGS. 1C and 1D). It is supposed, that the“closed” HER3 conformation can be opened via the binding of the ligandheregulin at a specific HER3 heregulin binding site. This takes place atthe HER3 interface formed by the HER3 ECD domains I and domain III. Bythis interaction it is believed, that the HER3 receptor is activated andtransferred into its “open conformation” (see FIGS. 1E and 1B and e.g.Baselga, J. et al, Nat Rev Cancer 9 (2009). 463-475 and Desbois-Mouthon,C., at al, Gastroenterol Clin Biol 34 (2010) 255-259). In this openconformation heterodimerization and transignal induction with HER2 ispossible (see FIG. 1B)

WO 2003/013602 relates to inhibitors of HER activity, including HERantibodies. WO 2007/077028 and WO 2008/100624 also relate to HER3antibodies.

WO 97/35885 and WO2010/127181 relate to HER3 antibodies.

Human HER4 (also known as ErbB-4 ERBB4, v-erb-a erythroblastic leukemiaviral oncogene homolog 4, p180erbB4 avian erythroblastic leukemia viral(v-erb-b2) oncogene homolog 4; SEQ ID NO:5) is a single-pass type Itransmembrane protein with multiple furin-like cysteine rich domains, atyrosine kinase domain, a phosphotidylinositol-3 kinase binding site anda PDZ domain binding motif (Plowman G D, wt al, PNAS 90:1746-50(1993);Zimonjic D B, et al, Oncogene 10:1235-7(1995); Culouscou J M, et al, J.Biol. Chem. 268:18407-10(1993)). The protein binds to and is activatedby neuregulins-2 and -3, heparin-binding EGF-like growth factor andbetacellulin. Ligand binding induces a variety of cellular responsesincluding mitogenesis and differentiation. Multiple proteolytic eventsallow for the release of a cytoplasmic fragment and an extracellularfragment. Mutations in this gene have been associated with cancer.Alternatively spliced variants which encode different protein isoformshave been described; however, not all variants have been fullycharacterized.

Anti-HER4 antibodies for use in anti-cancer therapy are known e.g. fromU.S. Pat. Nos. 5,811,098, 7,332,579 or Hollmén M, et al, Oncogene. 28(2009) 1309-19 (anti-ErbB-4 antibody mAb 1479).

So far it was not possible to select antigen binding proteins like e.g.antibodies that specifically bind to the beta-hairpin of HER3 and/orHER4 as these beta-hairpins of HER3 or of HER4 both represent hiddenepitopes, which are not accessible in the equilibrium state of thesereceptors (see FIGS. 1A-E).

SUMMARY OF THE INVENTION

We now have found a method using the beta-hairpins of HER3 and HER4functionally presented in a 3-dimensional orientation within SlyDscaffolds (see e.g. FIG. 2, and the polypeptides of SEQ ID NOs. 13, and17 to 24) to obtain such antigen binding proteins, in particularantibodies.

The invention provides a method for selecting an antigen bindingprotein, in particular an antibody that binds to human HER3 and binds tohuman HER4,

wherein the antigen binding protein, in particular the antibody, bindswithin an amino acid sequence of PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1) ofhuman HER3 and binds within an amino acid sequence ofPQTFVYNPTTFQLEHNFNA (SEQ ID NO:2) of human HER4;

wherein

-   -   a) at least one polypeptide selected from the group consisting        of:

SEQ ID NO: 13 TtSlyD-FKBP-Her3, SEQ ID NO: 17 TtSlyDcas-Her3, SEQ ID NO:18 TtSlyDcys-Her3, SEQ ID NO: 19 TgSlyDser-Her3, and SEQ ID NO: 20TgSlyDcys-Her3,

-   -   which comprises the amino acid sequence of SEQ ID NO:1;    -   and    -   b) at least one polypeptide selected from the group consisting        of:

SEQ ID NO: 21 TtSlyDcas-Her4, SEQ ID NO: 22 TtSlyDcys-Her4, SEQ ID NO:23 TgSlyDser-Her4, and SEQ ID NO: 24 TgSlyDcys-Her4,

-   -   which comprises the amino acid sequence of SEQ ID NO:2;

are used to select antigen binding proteins, in particular antibodies,which show binding to both, the at least one polypeptide under a) andthe at least one polypeptide under b)

and thereby selecting an antigen binding protein, in particular anantibody, that binds within an amino acid sequence ofPQPLVYNKLTFQLEPNPHT (SEQ ID NO:1) and within an amino acid sequence ofPQTFVYNPTTFQLEHNFNA (SEQ ID NO:2).

The invention provides antigen binding protein, in particular anantibody, obtained by such selection method.

The invention provides an isolated antigen binding protein, inparticular an antibody, that binds to human HER3 and binds to humanHER4, wherein the antigen binding protein, in particular the antibody,binds within an amino acid sequence of PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1)of human HER3 and binds within an amino acid sequence ofPQTFVYNPTTFQLEHNFNA (SEQ ID NO:2) of human HER4.

The invention provides an isolated antigen binding protein that binds tohuman HER3 and that binds to human HER4,

-   a) wherein the antigen binding protein binds to a polypeptide of

SEQ ID NO: 18 TtSlyDcys-Her3,

-   -   and

-   b) wherein the antigen binding protein binds to a polypeptide of

SEQ ID NO: 22 TtSlyDcys-Her4.

The invention further provides an isolated antigen binding protein thatbinds to human HER3 and that binds to human HER4,

-   a) wherein the antigen binding protein binds within an amino acid    sequence of PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1) which is comprised in    a polypeptide of SEQ ID NO: 18 (TtSlyDcys-Her3), and-   b) wherein the antigen binding protein binds within an amino acid    sequence of PQTFVYNPTTFQLEHNFNA (SEQ ID NO:2) which is comprised in    a polypeptide of SEQ ID NO: 22 (TtSlyDcys-Her4).

The invention provides an isolated antibody that binds to human HER3 andthat binds to human HER4,

-   a) wherein the antibody binds to a polypeptide of

SEQ ID NO: 18 TtSlyDcys-Her3,

-   -   and

-   b) wherein the antibody binds to a polypeptide of

SEQ ID NO: 22 TtSlyDcys-Her4.

The invention further provides an isolated antibody that binds to humanHER3 and that binds to human HER4,

-   a) wherein the antibody binds within an amino acid sequence of    PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1) which is comprised in a    polypeptide of SEQ ID NO: 18 (TtSlyDcys-Her3), and-   b) wherein the antibody binds within an amino acid sequence of    PQTFVYNPTTFQLEHNFNA (SEQ ID NO:2) which is comprised in a    polypeptide of SEQ ID NO: 22 (TtSlyDcys-Her4).

The invention provides an isolated antibody that binds to human HER3 andthat binds to human HER4, wherein the antibody

-   -   a) binds to the amino acid sequence of SEQ ID NO:1; and/or    -   b) binds to the amino acid sequence SEQ ID NO:1 in activated        HER3; and/or    -   c) binds within an amino acid sequence of PQPLVYNKLTFQLEPNPHT        (SEQ ID NO:1) which is comprised in a polypeptide selected from        the group consisting of:

SEQ ID NO: 13 TtSlyD-FKBP-Her3, SEQ ID NO: 17 TtSlyDcas-Her3, SEQ ID NO:18 TtSlyDcys-Her3, SEQ ID NO: 19 TgSlyDser-Her3, and SEQ ID NO: 20TgSlyDcys-Her3;

-   -    and/or    -   d) binds to the β-hairpin region of HER3; and/or    -   e) inhibits the heterodimerisation of HER3/HER2 heterodimers;        and/or    -   f) binds to HER3-ECD with a ratio of the association constant        (Ka) in presence of Heregulin (Ka (+Heregulin)) and in absence        of Heregulin (Ka (−Heregulin)) of 4.0 or higher (Ka        (+Heregulin))/(Ka (−Heregulin)); and/or    -   g) binds to HER3-ECD with a ratio of the Molar Ratio MR of        binding in presence of Heregulin (MR (+Heregulin)) and in        absence of Heregulin (MR (−Heregulin)) of 2.0 or higher (MR        (+Heregulin))/(MR (−Heregulin)); and/or    -   h) specifically binds to the amino acid sequence of SEQ ID NO:2;        and/or    -   i) binds to the amino acid sequence SEQ ID NO:2 in activated        HER4; and/or    -   j) binds within an amino acid sequence of PQTFVYNPTTFQLEHNFNA        (SEQ ID NO:2) which is comprised in a polypeptide selected from        the group consisting of:

SEQ ID NO: 21 TtSlyDcas-Her4, SEQ ID NO: 22 TtSlyDcys-Her4, SEQ ID NO:23 TgSlyDser-Her4, and SEQ ID NO: 24 TgSlyDcys-Her4;

-   -    and/or    -   k) binds to the β-hairpin region of HER4; and/or    -   l) binds to HER4-ECD with a ratio of the association constant        (Ka) in presence of Heregulin (Ka (+Heregulin)) and in absence        of Heregulin (Ka (−Heregulin)) of 20.0 or higher (Ka        (+Heregulin))/(Ka (−Heregulin)); and/or    -   m) binds to HER4-ECD with a ratio of the Molar Ratio MR of        binding in presence of Heregulin (MR (+Heregulin)) and in        absence of Heregulin (MR (−Heregulin)) of 5.0 or higher (MR        (+Heregulin))/(MR (−Heregulin)); and/or    -   n) shows as monovalent Fab fragment the same or higher        biological activity as compared to its bivalent parent full        length antibody; and/or    -   o) inhibits the HER3 phosporylation in MCF-7 cells; and/or    -   p) does not compete for binding to HER3 with Heregulin/induces        binding of Heregulin to HER3; and/or    -   q) inhibits the proliferation of MDA-MB-175 cells tumor cells;        and/or    -   r) shows tumor growth inhibitory activity in vivo; and/or    -   s) binds with an affinity of a KD value ≤1×10⁻⁸ M to HER3-ECD        (in one embodiment with a KD value of 1×10⁻⁸ M to 1×10⁻¹³ M; (in        one embodiment with a KD value of 1×10⁻⁹ M to 1×10⁻¹³ M); and/or    -   t) binds with an affinity of a KD value ≤1×10⁻⁸ M to HER4-ECD        (in one embodiment with a KD value of 1×10⁻⁸ M to 1×10⁻¹³ M; (in        one embodiment with a KD value of 1×10⁻⁹ M to 1×10⁻¹³ M); and/or    -   u) binds to a polypeptide consisting of VYNKLTFQLEP (SEQ ID        NO:43) and to a polypeptide of consisting of VYNPTTFQLE (SEQ ID        NO:44); and/or    -   v) binds to a polypeptide consisting of VYNKLTFQLEP (SEQ ID        NO:43); and/or    -   w) binds to a polypeptide consisting of VYNPTTFQLE (SEQ ID        NO:44); and/or    -   x) binds in a FACS assay to HER3 expressing T47D cells; and        wherein the antibody induces an at least 25% higher percentage        of internalization of HER3 in the presence of Heregulin as        compared to the percentage of internalization of HER3 in the        presence of Heregulin when measured after 1 h after antibody        exposure.

In one embodiment such anti-HER3/HER4 antibody is a monoclonal antibody.

In one embodiment such anti-HER3/HER4 antibody is a human, humanized, orchimeric antibody.

In one embodiment such anti-HER3/HER4 antibody is an antibody fragmentthat binds human HER3 and that binds human HER4.

In one embodiment such anti-HER3/HER4 antibody comprises (a) HVR-H1comprising the amino acid sequence of SEQ ID NO:25; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:26, and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:27.

In one embodiment such anti-HER3/HER4 antibody comprises (a) HVR-L1comprising the amino acid sequence of SEQ ID NO:28; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:29; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:30.

In one embodiment such anti-HER3/HER4 antibody comprises

-   -   i) (a) HVR-H1 comprising the amino acid sequence of SEQ ID        NO:25;    -   (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:26;    -   (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:27;    -   (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:28;    -   (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29;    -   and (f) HVR-L3 comprising the amino acid sequence of SEQ ID        NO:30;    -   ii) or a humanized variant of the HVRs of the antibody under i)        (a), (b), (d) and/or (e).

In one embodiment such anti-HER3/HER4 antibody comprises

-   -   i) (a) HVR-H1 comprising the amino acid sequence of SEQ ID        NO:38;    -   (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:39;    -   (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:27;    -   (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:40;    -   (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:41;    -   and (f) HVR-L3 comprising the amino acid sequence of SEQ ID        NO:30; or    -   ii) (a) HVR-H1 comprising the amino acid sequence of SEQ ID        NO:38;    -   (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:39;    -   (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:27;    -   (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:40;    -   (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:42;    -   and (f) HVR-L3 comprising the amino acid sequence of SEQ ID        NO:30; or    -   iii) (a) HVR-H1 comprising the amino acid sequence of SEQ ID        NO:25;    -   (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:39;    -   (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:27;    -   (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:40;    -   (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:41;    -   and (f) HVR-L3 comprising the amino acid sequence of SEQ ID        NO:30; or    -   iv) (a) HVR-H1 comprising the amino acid sequence of SEQ ID        NO:25;    -   (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:39;    -   (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:27;    -   (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:40;    -   (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:42;    -   and (f) HVR-L3 comprising the amino acid sequence of SEQ ID        NO:30.

In one embodiment such anti-HER3/HER4 antibody comprises (a) a VHsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:33; (b) a VL sequence having at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO:36; or (c) a VHsequence as in (a) and a VL sequence as in (b). In one embodiment suchanti-HER3/HER4 antibody comprises a VH sequence of SEQ ID NO: 33. In oneembodiment such anti-HER3/HER4 antibody comprises a VL sequence of SEQID NO: 36. In one embodiment such anti-HER3/HER4 antibody comprises a VHsequence of SEQ ID NO:33 and a VL sequence of SEQ ID NO:36.

In one embodiment such anti-HER3/HER4 antibody comprises (a) a VHsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:33; (b) a VL sequence having at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO:37; or (c) a VHsequence as in (a) and a VL sequence as in (b). In one embodiment suchanti-HER3/HER4 antibody comprises a VH sequence of SEQ ID NO: 33. In oneembodiment such anti-HER3/HER4 antibody comprises a VL sequence of SEQID NO: 37. In one embodiment such anti-HER3/HER4 antibody comprises a VHsequence of SEQ ID NO:33 and a VL sequence of SEQ ID NO:37.

In one embodiment such anti-HER3/HER4 antibody comprises (a) a VHsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:34; (b) a VL sequence having at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO:36; or (c) a VHsequence as in (a) and a VL sequence as in (b). In one embodiment suchanti-HER3/HER4 antibody comprises a VH sequence of SEQ ID NO: 34. In oneembodiment such anti-HER3/HER4 antibody comprises a VL sequence of SEQID NO: 36. In one embodiment such anti-HER3/HER4 antibody comprises a VHsequence of SEQ ID NO:34 and a VL sequence of SEQ ID NO:36.

In one embodiment such anti-HER3/HER4 antibody comprises (a) a VHsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:34; (b) a VL sequence having at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO:37; or (c) a VHsequence as in (a) and a VL sequence as in (b). In one embodiment suchanti-HER3/HER4 antibody comprises a VH sequence of SEQ ID NO: 34. In oneembodiment such anti-HER3/HER4 antibody comprises a VL sequence of SEQID NO: 37. In one embodiment such anti-HER3/HER4 antibody comprises a VHsequence of SEQ ID NO:34 and a VL sequence of SEQ ID NO:37.

In one embodiment such anti-HER3/HER4 antibody comprises (a) a VHsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:35; (b) a VL sequence having at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO:36; or (c) a VHsequence as in (a) and a VL sequence as in (b). In one embodiment suchanti-HER3/HER4 antibody comprises a VH sequence of SEQ ID NO: 35. In oneembodiment such anti-HER3/HER4 antibody comprises a VL sequence of SEQID NO: 36. In one embodiment such anti-HER3/HER4 antibody comprises a VHsequence of SEQ ID NO:35 and a VL sequence of SEQ ID NO:36.

In one embodiment such anti-HER3/HER4 antibody comprises (a) a VHsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:35; (b) a VL sequence having at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO:37; or (c) a VHsequence as in (a) and a VL sequence as in (b). In one embodiment suchanti-HER3/HER4 antibody comprises a VH sequence of SEQ ID NO: 35. In oneembodiment such anti-HER3/HER4 antibody comprises a VL sequence of SEQID NO: 37. In one embodiment such anti-HER3/HER4 antibody comprises a VHsequence of SEQ ID NO:35 and a VL sequence of SEQ ID NO:37.

In one embodiment such anti-HER3/HER4 antibody is a full length IgG1antibody or IgG4 antibody.

In one embodiment such anti-HER3/HER4 antibody is a Fab fragment.

The invention further provides an isolated nucleic acid suchanti-HER3/HER4 antibody.

The invention further provides a host cell comprising such nucleic acid.

The invention further provides a method of producing an antibodycomprising culturing such host cell so that the antibody is produced

In on embodiment such method further comprises recovering the antibodyfrom the host cell.

The invention further provides an immunoconjugate comprising suchanti-HER3/HER4 antibody and a cytotoxic agent.

The invention further provides a pharmaceutical formulation comprisingsuch anti-HER3/HER4 antibody and a pharmaceutically acceptable carrier.

The invention further provides the anti-HER3/HER4 antibody describedherein for use as a medicament. The invention further provides theanti-HER3/HER4 antibody described herein, or the immunoconjugatecomprising the anti-HER3/HER4 antibody and a cytotoxic agent, for use intreating cancer. The invention further provides the anti-HER3/HER4antibody described herein for use in inhibition of HER3/HER2dimerization.

Use of such anti-HER3/HER4 antibody, or an immunoconjugate comprisingthe anti-HER3/HER4 antibody and a cytotoxic agent, in the manufacture ofa medicament. Such use wherein the medicament is for treatment ofcancer. Such use wherein the medicament is for the inhibition ofHER3/HER2 dimerization.

The invention further provides a method of treating an individual havingcancer comprising administering to the individual an effective amount ofthe anti-HER3/HER4 antibody described herein, or an immunoconjugatecomprising the anti-HER3/HER4 antibody and a cytotoxic agent.

The invention further provides a method of inducing apoptosis in acancer cell in an individual suffering from cancer comprisingadministering to the individual an effective amount of animmunoconjugate comprising the anti-HER3/HER4 antibody described hereinand a cytotoxic agent, thereby inducing apoptosis in a cancer cell inthe individual.

One embodiment of the invention is a polypeptide selected from the groupconsisting of:

i) SEQ ID NO: 13 TtSlyD-FKBP-Her3, ii) SEQ ID NO: 17 TtSlyDcas-Her3,iii) SEQ ID NO: 18 TtSlyDcys-Her3, iv) SEQ ID NO: 19 TgSlyDser-Her3, andv) SEQ ID NO: 20 TgSlyDcys-Her3,

which polypeptide comprises the amino acid sequence of SEQ ID NO:1

One embodiment of the invention is a polypeptide selected from the groupconsisting of:

i) SEQ ID NO: 21 TtSlyDcas-Her4, ii) SEQ ID NO: 22 TtSlyDcys-Her4, iii)SEQ ID NO: 23 TgSlyDser-Her4, and iv) SEQ ID NO: 24 TgSlyDcys-Her4,

which polypeptide comprises the amino acid sequence of SEQ ID NO:2.

The invention further provides the use of one of such polypeptidesselected from the group consisting of:

i) SEQ ID NO: 13 TtSlyD-FKBP-Her3, ii) SEQ ID NO: 17 TtSlyDcas-Her3,iii) SEQ ID NO: 18 TtSlyDcys-Her3, iv) SEQ ID NO: 19 TgSlyDser-Her3, andv) SEQ ID NO: 20 TgSlyDcys-Her3,

for eliciting an immune response against SEQ ID NO:1 in an experimentalanimal.

The invention further provides the use of one of such polypeptidesselected from the group consisting of:

i) SEQ ID NO: 21 TtSlyDcas-Her4, ii) SEQ ID NO: 22 TtSlyDcys-Her4, iii)SEQ ID NO: 23 TgSlyDser-Her4, and iv) SEQ ID NO: 24 TgSlyDcys-Her4,

for eliciting an immune response against SEQ ID NO:2 in an experimentalanimal.

The invention further provides a method for producing an antibodyspecifically binding to the β-hairpin of HER3 with the amino acidsequence of SEQ ID NO:1 comprising the following steps:

-   a) administering to an experimental animal a polypeptide selected    from the group consisting of:

i) SEQ ID NO: 13 TtSlyD-FKBP-Her3, ii) SEQ ID NO: 17 TtSlyDcas-Her3,iii) SEQ ID NO: 18 TtSlyDcys-Her3, iv) SEQ ID NO: 19 TgSlyDser-Her3, andv) SEQ ID NO: 20 TgSlyDcys-Her3,

for at least one time, whereby the polypeptide comprises the β-hairpinof HER3 with the amino acid sequence of SEQ ID NO:1,

-   b) recovering from the experimental animal three to ten days after    the last administration of the polypeptide B-cells that produce the    antibody specifically binding to the β-hairpin of HER3 with the    amino acid sequence of SEQ ID NO:1, and-   c) cultivating a cell comprising a nucleic acid encoding the    antibody specifically binding to the the β-hairpin of HER3 with the    amino acid sequence of SEQ ID NO:1 and recovering the antibody from    the cell or the cultivation medium and thereby producing an antibody    specifically binding to a target antigen.

The invention further provides the use of a polypeptide selected fromthe group consisting of:

i) SEQ ID NO: 13 TtSlyD-FKBP-Her3, ii) SEQ ID NO: 17 TtSlyDcas-Her3,iii) SEQ ID NO: 18 TtSlyDcys-Her3, iv) SEQ ID NO: 19 TgSlyDser-Her3, andv) SEQ ID NO: 20 TgSlyDcys-Her3,

for epitope mapping, whereby the polypeptide comprises the epitope inthe the β-hairpin of HER3 with the amino acid sequence of SEQ ID NO:1.

A method for producing an antibody specifically binding to the β-hairpinof HER4 with the amino acid sequence of SEQ ID NO:2 comprising thefollowing steps:

-   a) administering to an experimental animal a polypeptide selected    from the group consisting of:

i) SEQ ID NO: 21 TtSlyDcas-Her4, ii) SEQ ID NO: 22 TtSlyDcys-Her4, iii)SEQ ID NO: 23 TgSlyDser-Her4, and iv) SEQ ID NO: 24 TgSlyDcys-Her4,

for at least one time, whereby the polypeptide comprises the β-hairpinof HER4 with the amino acid sequence of SEQ ID NO:2,

-   b) recovering from the experimental animal three to ten days after    the last administration of the polypeptide B-cells that produce the    antibody specifically binding to the β-hairpin of HER4 with the    amino acid sequence of SEQ ID NO:2, and-   c) cultivating a cell comprising a nucleic acid encoding the    antibody specifically binding to the the β-hairpin of HER4 with the    amino acid sequence of SEQ ID NO:2 and recovering the antibody from    the cell or the cultivation medium and thereby producing an antibody    specifically binding to a target antigen.

The invention further provides the use of a polypeptide selected fromthe group consisting of:

i) SEQ ID NO: 21 TtSlyDcas-Her4, ii) SEQ ID NO: 22 TtSlyDcys-Her4, iii)SEQ ID NO: 23 TgSlyDser-Her4, and iv) SEQ ID NO: 24 TgSlyDcys-Her4,

for epitope mapping, whereby the polypeptide comprises the epitope inthe the β-hairpin of HER4 with the amino acid sequence of SEQ ID NO:2.

Using the beta-hairpins of HER3 and HER4 functionally presented in a3-dimensional orientation within SlyD scaffolds (see e.g. FIG. 2, andthe polypeptides of SEQ ID NOs. 13, and 17 to 24) the anti-HER3/HER4antigen binding proteins, in particular antibodies, described hereinbinding to these beta-hairpins could be selected. It was found that theantigen binding proteins, in particular antibodies,according to theinvention have highly valuable properties such as strong growthinhibition of HER3 expressing cancer cells, strong inhibition of HER3mediated signal transduction (such as e.g. HER3 phoshorylation) which isrelated to cancer cell proliferation, or very specific pharmacokineticproperties (such as faster association rates and higher Molar Ratios ofthe binding the activated HER3 in the presence of Heregulin (“openconformation) when compared to the absence of Heregulin (“closedconformation”).

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-E Schematic overview of “closed” and “open” HER3 conformationand the influence of the Neuregulin family ligands (like e.g. Heregulinabbreviated here as HR) on the conformation change.

FIG. 2 3D-structure of the beta-hairpin of HER3 functionally presentedin a 3-dimensional orientation within a SlyD scaffold of Thermusthermophiles.

FIG. 3 SDS-PAGE analysis of Ni-NTA purification of TtSlyD-FKBP-Her3. E1and E2 show the purified fractions 12 and 13.SN: E. coli lysatesupernatant before purification.

FIG. 4 SEC elution profile of a Ni-NTA purified fraction of Thermusthermophilus SlyD-FKBP-Her-3.

FIG. 5 Testing of specificity and reactivity in IHC of the selectedclones. All three clones showed binding to Her3 and cross reactivityagainst Her4. No cross reactivity against Her1 and Her2 was detectable.

FIG. 6 FACS analysis of M-05-74 antibody induced time dependent HER3internalization in T47D cells.

FIG. 7 Biacore sensorgram overlay plot. 1: 100 nMM-05-74*Heregulin/Her-3 ECD interaction. 2: 100 nMM-08-11*Heregulin/Her-3 ECD interaction. 3&4: 100 nM M-05-74 and 100 nMM-08-11*Her-3 ECD interaction. 5: buffer reference.

FIG. 8 Sensorgram overlay of the Biacore epitope-binning experiment. Theprimary antibody M-05-74 (M-074 in the Figure) presented the Her-3 ECDto the secondary antibodies M-208, GT (=8B8), M-05-74 and M-08-11 (M-011in the FIG. 8) (M-. The noise of the measurement was 5 RU.

FIG. 9 Biacore sensorgram overlay plot. 1: 90 nM Heregulin*Her-3 ECDcomplex on M-05-74. 2: 90 nM Heregulin*Her-3 ECD complex on M-08-11. 3:90 nM Heregulin*Her-3 ECD complex on 8B8 antibody.

FIG. 10 Schematic Mode of Actions identified by Biacore functionalassays. 1: M-08-11 binds to the Heregulin activated Her-3 ECD andinduces a delayed Heregulin dissociation, whereby M-08-11 stays in theHer-3 ECD receptor complex. 2: M-05-74 binds to the Heregulin activatedHer-3 ECD. Heregulin is trapped in the complex and the antibody stays inthe complex 3: 8B8 binds the Heregulin activated Her-3 ECD. The wholecomplex dissociates from the antibody.

FIG. 11 Strategy of the epitope mapping and alanine-scan approach. Thepeptide hairpin sequences (peptide hairpin) of EGFR, Her-2 ECD, Her-3ECD and Her-4 ECD including their structural embeddings (structural)were investigated. Cysteins were replaced by serines.

FIG. 12 CelluSpots™ Synthesis and Epitope Mapping of epitopes ofantibody M-05-74 on HER3 and HER4. Anti-HER3/HER4 antibody M-05-74 bindsto HER3 ECD binding epitope VYNKLTFQLEP (SEQ ID NO:43) and to HER4 ECDbinding epitope VYNPTTFQLE (SEQ ID NO:44).

FIG. 13 Results from the CelluSpots™ Ala-Scan of anti HER3/HER4 antibodyM-05-74 (named M-074 in the Figure) and anti-HER3 antibody M-08-11(named M-011) with no HER4 crossreactivity)—the amino acids which arecontributing most to the binding of anti-HER3/HER4 antibody M-05-74 toits HER3 ECD binding epitope VYNKLTFQLEP (SEQ ID NO:43) and to its HER4ECD binding epitope VYNPTTFQLE (SEQ ID NO:44) are underlined/bold.

FIG. 14 Binding of M-05-74 (M-074) induces/promotes binding of HRG tothe HER3-ECD.

FIG. 15 Inhibition of HER2/HER3 heterodimers/heterodimerization(Imunoprecipitation and Western Blot) in MCF7 cells(HER3-IP=immunoprecipitation with HER3antibody/HER2-IP=immunoprecipitation with HER3 antibody).

FIG. 16 Treatment of MDA-MB175 cells with M-05-74 resulted in inhibitionof cell proliferation.

FIG. 17 Treatment with M-05-74 (M-074) (10 mg/kg q7d, i.p.) resulted intumor stasis a FaDu HNSCC transplanted xenografts.

FIG. 18 Treatment with M-05-74-Fab-Pseudomonas exotoxin conjugate(M-074-PE) (10 mg/kg q7d, i.p.) resulted in stronger inhibition of cellproliferation in the presence (bold line) of HRG than in the absence(thin line) of HRG.

FIG. 19 In vivo tumor cell growth inhibition by M-05-74-Fab-Pseudomonasexotoxin conjugate (M-05-74-PE). Legend: closed line (vehicle); dottedline (M-05-74-Fab-Pseudomonas exotoxin conjugate (M-05-74-PE)).

FIG. 20 Biacore sensorgram overlay plot: binding of the antibody M-05-74(1) of the present invention to TtSlyDcys-Her3 (SEQ ID NO: 18) incomparison with anti-HER3 antibody MOR09823 (2) described inWO2012/22814. While the antibody of the present M-05-74 (1) shows aclear binding signal to TtSlyDcys-Her3 (SEQ ID NO: 18), the antibodyanti-HER3 antibody MOR09823 (2) shows no binding at all toTtSlyDcys-Her3 (SEQ ID NO: 18). Control measurement (3) without antibodyat all did not shown any binding to TtSlyDcys-Her3 (SEQ ID NO: 18).

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

I. Definitions

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs), compared to aparent antibody which does not possess such alterations, suchalterations resulting in an improvement in the affinity of the antibodyfor antigen.

The term “antigen binding protein” as used herein refers to an antibodyas described herein or to a scaffold antigen binding protein. In onepreferred embodiment the antigen binding protein is an antibody asdescribed herein. Scaffold antigen binding proteins are known in theart, for example, fibronectin and designed ankyrin-repeat proteins(DARPins) have been used as alternative scaffolds for antigen-bindingdomains, see, e.g., Gebauer and Skerra, Engineered protein scaffolds asnext-generation antibody therapeutics. Curr Opin Chem Biol 13:245-255(2009) and Stumpp et al., Darpins: A new generation of proteintherapeutics. Drug Discov Today 13: 695-701 (2008), both of which areincorporated herein by reference in their entirety. B. Criteria forSelecting Parent Variable Domains and Receptors for antigen bindingproteins of the invention. In one embodiment a scaffold antigen bindingprotein is selected from the group consisting of CTLA-4 (Evibody);lipocalin; Protein A derived molecules such as Z-domain of Protein A(Affibody, SpA), A-domain (Avimer/Maxibody); Heat shock proteins such asGroEI and GroES; transferrin (trans-body); ankyrin repeat protein(DARPin); peptide aptamer; C-type lectin domain (Tetranectin); human.gamma.-crystallin and human ubiquitin (affilins); PDZ domains; scorpiontoxinkunitz type domains of human protease inhibitors; and fibronectin(adnectin); which has been subjected to protein engineering in order toobtain binding to a ligand other than the natural ligand.

CTLA-4 (Cytotoxic T Lymphocyte-associated Antigen 4) is a CD28-familyreceptor expressed on mainly CD4+T-cells. Its extracellular domain has avariable domain-like Ig fold. Loops corresponding to CDRs of antibodiescan be substituted with heterologous sequence to confer differentbinding properties. CTLA-4 molecules engineered to have differentbinding specificities are also known as Evibodies. For further detailssee Journal of Immunological Methods 248 (1-2), 31-45 (2001).

Lipocalins are a family of extracellular proteins which transport smallhydrophobic molecules such as steroids, bilins, retinoids and lipids.They have a rigid .beta.-sheet secondary structure with a number ofloops at the open end of the conical structure which can be engineeredto bind to different target antigens. Anticalins are between 160-180amino acids in size, and are derived from lipocalins. For furtherdetails see Biochim Biophys Acta 1482: 337-350 (2000), U.S. Pat. No.7,250,297 B1 and US 2007/0224633.

An affibody is a scaffold derived from Protein A of Staphylococcusaureus which can be engineered to bind to antigen. The domain consistsof a three-helical bundle of approximately 58 amino acids. Librarieshave been generated by randomisation of surface residues. For furtherdetails see Protein Eng. Des. SeI. 17, 455-462 (2004) andEP1641818A1Avimers are multidomain proteins derived from the A-domainscaffold family. The native domains of approximately 35 amino acidsadopt a defined disulphide bonded structure. Diversity is generated byshuffling of the natural variation exhibited by the family of A-domains.For further details see Nature Biotechnology 23(12), 1556-1561 (2005)and Expert Opinion on Investigational Drugs 16(6), 909-917 (June 2007).

A transferrin is a monomeric serum transport glycoprotein. Transferrinscan be engineered to bind different target antigens by insertion ofpeptide sequences in a permissive surface loop. Examples of engineeredtransferrin scaffolds include the Trans-body. For further details see J.Biol. Chem 274, 24066-24073 (1999).

Designed Ankyrin Repeat Proteins (DARPins) are derived from Ankyrinwhich is a family of proteins that mediate attachment of integralmembrane proteins to the cytoskeleton. A single ankyrin repeat is a 33residue motif consisting of two .alpha.-helices and a .beta.-turn. Theycan be engineered to bind different target antigens by randomisingresidues in the first .alpha.-helix and a .beta.-turn of each repeat.Their binding interface can be increased by increasing the number ofmodules (a method of affinity maturation). For further details see J.MoI. Biol. 332, 489-503 (2003), PNAS 100(4), 1700-1705 (2003) and J.MoI. Biol. 369, 1015-1028 (2007) and US20040132028A1.

Fibronectin is a scaffold which can be engineered to bind to antigen.Adnectins consists of a backbone of the natural amino acid sequence ofthe 10th domain of the 15 repeating units of human fibronectin type III(FN3). Three loops at one end of the .beta.-sandwich can be engineeredto enable an Adnectin to specifically recognize a therapeutic target ofinterest. For further details see Protein Eng. Des. SeI. 18, 435-444(2005), US20080139791, WO2005056764 and U.S. Pat. No. 6,818,418B1.

Peptide aptamers are combinatorial recognition molecules that consist ofa constant scaffold protein, typically thioredoxin (TrxA) which containsa constrained variable peptide loop inserted at the active site. Forfurther details see Expert Opin. Biol. Ther. 5, 783-797 (2005).

Microbodies are derived from naturally occurring microproteins of 25-50amino acids in length which contain 3-4 cysteine bridges—examples ofmicroproteins include KalataBI and conotoxin and knottins. Themicroproteins have a loop which can be engineered to include up to 25amino acids without affecting the overall fold of the microprotein. Forfurther details of engineered knottin domains, see WO2008098796.

Other antigen binding proteins include proteins which have been used asa scaffold to engineer different target antigen binding propertiesinclude human .gamma.-crystallin and human ubiquitin (affilins), kunitztype domains of human protease inhibitors, PDZ-domains of theRas-binding protein AF-6, scorpion toxins (charybdotoxin), C-type lectindomain (tetranectins) are reviewed in Chapter 7—Non-Antibody Scaffoldsfrom Handbook of Therapeutic Antibodies (2007, edited by Stefan Dubel)and Protein Science 15:14-27 (2006). Epitope binding domains of thepresent invention could be derived from any of these alternative proteindomains.

The terms “anti-HER3/HER4 antigen binding protein”, “an antigen bindingprotein that binds to (human) HER3 and binds to (human) HER4” and “anantigen binding protein that binds specifically to human HER3 andspecifically binds to human HER4” refer to an antigen binding proteinthat is capable of binding HER3 or HER4 with sufficient affinity suchthat the antibody is useful as a diagnostic and/or therapeutic agent intargeting HER3 and/or HER4.

The terms “anti-HER3/HER4 antibody”, “an antibody that binds to (human)HER3 and binds to (human) HER4” and “an antibody that binds specificallyto human HER3 and specifically binds to human HER4” refer to an antibodythat is capable of binding HER3 or HER4 with sufficient affinity suchthat the antibody is useful as a diagnostic and/or therapeutic agent intargeting HER3 and/or HER4. In one embodiment, the extent of binding ofan anti-HER3/HER4 antibody to an unrelated, non-HER3/HER4 protein isless than about 10% of the binding of the antibody to HER3 or HER4 asmeasured, e.g., by a Surface Plasmon Resonance assay (e.g. BIACORE). Incertain embodiments, an antibody that binds to human HER3 has a KD valueof the binding affinity for binding to human HER3 of ≤1 μM, ≤100 nM, ≤10nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸ M or less, e.g.from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M). In certainembodiments, an antibody that binds to human HER4 has a KD value of thebinding affinity for binding to human HER4 of ≤1 μM, ≤100 nM, ≤10 nM, ≤1nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10-8 M or less, e.g. from 10-8M to 10-13 M, e.g., from 10-9 M to 10-13 M).b In one preferredembodiment the respective KD value of the binding affinities isdetermined in a Surface Plasmon Resonance assay using the wildtypeExtracellular domain (ECD) of human HER3 (HER3-ECD) for the HER3 bindingaffinity, and wildtype human HER4-ECD for the HER4 binding affinity,respectively.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)2; diabodies; linear antibodies; single-chain antibody molecules(e.g. scFv); and multispecific antibodies formed from antibodyfragments.

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that blocks binding of the reference antibody toits antigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. An exemplary competition assay isprovided herein.

The term “cancer” as used herein may be, for example, lung cancer, nonsmall cell lung (NSCL) cancer, bronchioloalviolar cell lung cancer, bonecancer, pancreatic cancer, skin cancer, cancer of the head or neck,cutaneous or intraocular melanoma, uterine cancer, ovarian cancer,rectal cancer, cancer of the anal region, stomach cancer, gastriccancer, colon cancer, breast cancer, uterine cancer, carcinoma of thefallopian tubes, carcinoma of the endometrium, carcinoma of the cervix,carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease,cancer of the esophagus, cancer of the small intestine, cancer of theendocrine system, cancer of the thyroid gland, cancer of the parathyroidgland, cancer of the adrenal gland, sarcoma of soft tissue, cancer ofthe urethra, cancer of the penis, prostate cancer, cancer of thebladder, cancer of the kidney or ureter, renal cell carcinoma, carcinomaof the renal pelvis, mesothelioma, hepatocellular cancer, biliarycancer, neoplasms of the central nervous system (CNS), spinal axistumors, brain stem glioma, glioblastoma multiforme, astrocytomas,schwanomas, ependymonas, medulloblastomas, meningiomas, squamous cellcarcinomas, pituitary adenoma, lymphoma, lymphocytic leukemia, includingrefractory versions of any of the above cancers, or a combination of oneor more of the above cancers. In one preferred embodiment such cancer isa breast cancer, ovarian cancer, cervical cancer, lung cancer orprostate cancer. In one preferred embodiment such cancers are furthercharacterized by HER3 and/or HER4 expression or overexpression. Onefurther embodiment the invention are the anti-HER3/HER4 antibodies ofthe present invention for use in the simultaneous treatment of primarytumors and new metastases.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ respectively.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153,Bi212, P32, Pb212 and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof; and the variousantitumor or anticancer agents disclosed below. In one preferredembodiment the “cytotoxic agent” is Pseudomonas exotoxin A or variantsthereof. In one preferred embodiment the “cytotoxic agent” is amatoxinor a variants thereof.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

The term “epitope” includes any polypeptide determinant capable ofspecific binding to an antibody. In certain embodiments, epitopedeterminant include chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, incertain embodiments, may have specific three dimensional structuralcharacteristics, and or specific charge characteristics. An epitope is aregion of an antigen that is bound by an antibody.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat, E. A. et al., Sequences of Proteins of Immunological Interest,5th ed., Public Health Service, National Institutes of Health, Bethesda,Md. (1991), NIH Publication 91-3242.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3 (L3)-FR4.

The terms “full length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat, E. A. et al., Sequences of Proteins of Immunological Interest,5th ed., Bethesda Md. (1991), NIH Publication 91-3242, Vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized variant” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization. In one preferred embodiment, a murine HVR is grafted intothe framework region of a human antibody to prepare the “humanizedantibody.” See e.g. Riechmann, L., et al., Nature 332 (1988) 323-327;and Neuberger, M. S., et al., Nature 314 (1985) 268-270. The murinevariable region amino acid sequence is aligned to a collection of humangermline antibody V-genes, and sorted according to sequence identity andhomology. The acceptor sequence is selected based on high overallsequence homology and optionally also the presence of the rightcanonical residues already in the acceptor sequence (see Poul, M-A. andLefranc, M-P., in “Ingénierie des anticorps banques combinatores” ed. byLefranc, M-P. and Lefranc, G., Les Editions INSERM, 1997). The germlineV-gene encodes only the region up to the beginning of HVR3 for the heavychain, and till the middle of HVR3 of the light chain. Therefore, thegenes of the germline V-genes are not aligned over the whole V-domain.The humanized construct comprises the human frameworks 1 to 3, themurine HVRs, and the human framework 4 sequence derived from the humanJK4, and the JH4 sequences for light and heavy chain, respectively.Before selecting one particular acceptor sequence, the so-calledcanonical loop structures of the donor antibody can be determined (seeMorea, V., et al., Methods, Vol 20, Issue 3 (2000) 267-279). Thesecanonical loop structures are determined by the type of residues presentat the so-called canonical positions. These positions lie (partially)outside of the HVR regions, and should be kept functionally equivalentin the final construct in order to retain the HVR conformation of theparental (donor) antibody. In WO 2004/006955 a method for humanizingantibodies is reported that comprises the steps of identifying thecanonical HVR structure types of the HVRs in a non-human matureantibody; obtaining a library of peptide sequence for human antibodyvariable regions; determining the canonical HVR structure types of thevariable regions in the library; and selecting the human sequences inwhich the canonical HVR structure is the same as the non-human antibodycanonical HVR structure type at corresponding locations within thenon-human and human variable regions. Summarizing, the potentialacceptor sequence is selected based on high overall homology andoptionally in addition the presence of the right canonical residuesalready in the acceptor sequence. In some cases simple HVR grafting onlyresult in partial retention of the binding specificity of the non-humanantibody. It has been found that at least some specific non-humanframework residues are required for reconstituting the bindingspecificity and have also to be grafted into the human framework, i.e.so called “back mutations” have to be made in addition to theintroduction of the non-human HVRs (see e.g. Queen et al., Proc. Natl.Acad. Sci. USA 86 (1989) 10,029-10,033; Co et al., Nature 351 (1991)501-502). These specific framework amino acid residues participate inFR-HVR interactions and stabilized the conformation (loop) of the HVRs(see e.g. Kabat et al., J. Immunol. 147 (1991) 1709). In some cases alsoforward-mutations are introduced in order to adopt more closely thehuman germline sequence. Thus “humanized variant of an antibodyaccording to the invention” (which is e.g. of mouse origin) refers to anantibody, which is based on the mouse antibody sequences in which the VHand VL are humanized by above described standard techniques (includingHVR grafting and optionally subsequent mutagenesis of certain aminoacids in the framework region and the HVR-H1, HVR-H2, HVR-L1 or HVR-L2,whereas HVR-H3 and HVR-L3 remain unmodified).

The term “hypervariable region” or “HVR” as used herein refers to eachof the regions of an antibody variable domain which are hypervariable insequence (“complementarity determining regions” or “CDRs”) and/or formstructurally defined loops (“hypervariable loops”) and/or contain theantigen-contacting residues (“antigen contacts”). Generally, antibodiescomprise six HVRs: three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). Exemplary HVRs herein include:

-   (a) hypervariable loops occurring at amino acid residues 26-32 (L1),    50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3)    (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));-   (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2),    89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al.,    Sequences of Proteins of Immunological Interest, 5th Ed. Public    Health Service, National Institutes of Health, Bethesda, Md.    (1991));-   (c) antigen contacts occurring at amino acid residues 27c-36 (L1),    46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3)    (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and-   (d) combinations of (a), (b), and/or (c), including HVR amino acid    residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1),    26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

Unless otherwise indicated, HVR residues and other residues in thevariable domain (e.g., FR residues) are numbered herein according toKabat et al., supra.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman, S. et al., J. Chromatogr. B 848 (2007) 79-87.

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding an anti-HER3/HER4 antibody” refers toone or more nucleic acid molecules encoding antibody heavy and lightchains (or fragments thereof), including such nucleic acid molecule(s)in a single vector or separate vectors, and such nucleic acidmolecule(s) present at one or more locations in a host cell.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

The term “Mab” refers to monoclonal antibodies, whereas the term “hMab”refers to humanized variants of such monoclonal antibodies.

A “naked antibody” refers to an antibody that is not conjugated to aheterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The nakedantibody may be present in a pharmaceutical formulation. (Include ifPrior art has immunoconjugates).

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

The term “HER3,” as used herein, refers to any native HER3 from anyvertebrate source, including mammals such as primates (e.g. humans) androdents (e.g., mice and rats), unless otherwise indicated. The termencompasses “full-length,” unprocessed HER3 as well as any form of HER3that results from processing in the cell. The term also encompassesnaturally occurring variants of HER3, e.g., splice variants or allelicvariants. The amino acid sequence of an exemplary human HER3 is shown inSEQ ID NO:3. “Human HER3” (ErbB-3, ERBB3, c-erbB-3,c-erbB3, receptortyrosine-protein kinase erbB-3, SEQ ID NO: 3) encodes a member of theepidermal growth factor receptor (EGFR) family of receptor tyrosinekinases which also includes HER1 (also known as EGFR), HER2, and HER4(Kraus, M. H. et al, PNAS 86 (1989) 9193-9197; Plowman, G. D. et al,PNAS 87 (1990) 4905-4909; Kraus, M. H. et al, PNAS 90 (1993) 2900-2904).Like the prototypical epidermal growth factor receptor, thetransmembrane receptor HER3 consists of an extracellular ligand-bindingdomain (ECD), a dimerization domain within the ECD, a transmembranedomain, an intracellular protein tyrosine kinase domain (TKD) and aC-terminal phosphorylation domain. This membrane-bound protein has aHeregulin (HRG) binding domain within the extracellular domain but notan active kinase domain. It therefore can bind this ligand but notconvey the signal into the cell through protein phosphorylation.However, it does form heterodimers with other HER family members whichdo have kinase activity. Heterodimerization leads to the activation ofthe receptor-mediated signaling pathway and transphosphorylation of itsintracellular domain. Dimer formation between HER family members expandsthe signaling potential of HER3 and is a means not only for signaldiversification but also signal amplification. For example the HER2/HER3heterodimer induces one of the most important mitogenic signals via thePI3K and AKT pathway among HER family members (Sliwkowski M. X., et al,J. Biol. Chem. 269 (1994) 14661-14665; Alimandi M, et al, Oncogene. 10(1995) 1813-1821; Hellyer, N. J., J. Biol. Chem. 276 (2001) 42153-4261;Singer, E., J. Biol. Chem. 276 (2001) 44266-44274; Schaefer, K. L.,Neoplasia 8 (2006) 613-622) For an overview of HER3 and its variousinteractions within the HER receptor family and the NGR ligands familysee e.g. G Sithanandam et al Cancer Gene Therapy (2008) 15, 413-448.

Interestingly in its equilibrium state, the HER3 receptors exists in its“closed confirmation”, which does mean, the heterodimerization HER3beta-hairpin motive is tethered via non-covalent interactions to theHER3 ECD domain IV (see FIG. 1C). It is supposed, that the “closed” HER3conformation can be opened via the binding of the ligand heregulin at aspecific HER3 heregulin binding site. This takes place at the HER3interface formed by the HER3 ECD domains I and domain III. By thisinteraction it is believed, that the HER3 receptor is activated andtransferred into its “open conformation” (see FIG. 1B and e.g. Baselga,J. et al, Nat Rev Cancer 9 (2009). 463-475 and Desbois-Mouthon, C., atal, Gastroenterol Clin Biol 34 (2010) 255-259). In this openconformation heterodimerization and transignal induction with HER2 ispossible (see FIG. 1B).

The term “HER4,” as used herein, refers to any native HER4 from anyvertebrate source, including mammals such as primates (e.g. humans) androdents (e.g., mice and rats), unless otherwise indicated. The termencompasses “full-length,” unprocessed HER4 as well as any form of HER4that results from processing in the cell. The term also encompassesnaturally occurring variants of HER4, e.g., splice variants or allelicvariants. The amino acid sequence of an exemplary human HER4 is shown inSEQ ID NO:5. “Human HER4” (also known as ErbB-4 ERBB4, v-erb-aerythroblastic leukemia viral oncogene homolog 4, p180erbB4 avianerythroblastic leukemia viral (v-erb-b2) oncogene homolog 4; SEQ IDNO:5) is a single-pass type I transmembrane protein with multiplefurin-like cysteine rich domains, a tyrosine kinase domain, aphosphotidylinositol-3 kinase binding site and a PDZ domain bindingmotif (Plowman G D, wt al, PNAS 90:1746-50(1993); Zimonjic D B, et al,Oncogene 10:1235-7(1995); Culouscou J M, et al, J. Biol. Chem.268:18407-10(1993)). The protein binds to and is activated byneuregulins-2 and -3, heparin-binding EGF-like growth factor andbetacellulin. Ligand binding induces a variety of cellular responsesincluding mitogenesis and differentiation. Multiple proteolytic eventsallow for the release of a cytoplasmic fragment and an extracellularfragment. Mutations in this gene have been associated with cancer.Alternatively spliced variants which encode different protein isoformshave been described; however, not all variants have been fullycharacterized.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies ofthe invention are used to delay development of a disease or to slow theprogression of a disease.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt,T. J. et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., N.Y.(2007), page 91) A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See, e.g., Portolano, S. et al., J.Immunol. 150 (1993) 880-887; Clackson, T. et al., Nature 352 (1991)624-628).

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors”.

II. Compositions and Methods

In one aspect, the invention is based, in part, on the finding thatusing the beta-hairpins of HER3 and HER4 functionally presented in a3-dimensional orientation within SlyD scaffolds (see e.g. FIG. 2, andthe polypeptides of SEQ ID NO. 13, and 17 to 24) it was possible toselect antibodies which are specific for both, the beta-hairpin of HER3and HER4.

In certain embodiments, the invention provides an antibody that binds tohuman HER3 and binds to human HER4, wherein the antibody binds within anamino acid sequence of PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1) of human HER3and binds within an amino acid sequence of PQTFVYNPTTFQLEHNFNA (SEQ IDNO:2) of human HER4.

Antibodies of the invention are useful, e.g., for the diagnosis ortreatment of cancer.

A. Exemplary Anti-HER3/HER4 Antigen Binding Proteins and Antibodies

The invention provides an isolated antigen binding protein that binds tohuman HER3 and that binds to human HER4,

-   a) wherein the antigen binding protein binds to a polypeptide    selected from the group consisting of:

SEQ ID NO: 13 TtSlyD-FKBP-Her3, SEQ ID NO: 17 TtSlyDcas-Her3, SEQ ID NO:18 TtSlyDcys-Her3, SEQ ID NO: 19 TgSlyDser-Her3, and SEQ ID NO: 20TgSlyDcys-Her3,

-   -   and

-   b) wherein the antigen binding protein binds to a polypeptide    selected from the group consisting of:

SEQ ID NO: 21 TtSlyDcas-Her4, SEQ ID NO: 22 TtSlyDcys-Her4, SEQ ID NO:23 TgSlyDser-Her4, and SEQ ID NO: 24 TgSlyDcys-Her4.

The invention further provides an isolated antigen binding protein thatbinds to human HER3 and that binds to human HER4,

-   a) wherein the antigen binding protein binds within an amino acid    sequence of PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1) which is comprised in    a polypeptide selected from the group consisting of:

SEQ ID NO: 13 TtSlyD-FKBP-Her3, SEQ ID NO: 17 TtSlyDcas-Her3, SEQ ID NO:18 TtSlyDcys-Her3, SEQ ID NO: 19 TgSlyDser-Her3, and SEQ ID NO: 20TgSlyDcys-Her3;

-   -   and

-   b) wherein the antigen binding protein binds within an amino acid    sequence of PQTFVYNPTTFQLEHNFNA (SEQ ID NO:2) which is comprised in    a polypeptide selected from the group consisting of:

SEQ ID NO: 21 TtSlyDcas-Her4, SEQ ID NO: 22 TtSlyDcys-Her4, SEQ ID NO:23 TgSlyDser-Her4, and SEQ ID NO: 24 TgSlyDcys-Her4.

The invention provides an isolated antigen binding protein that binds tohuman HER3 and that binds to human HER4,

-   a) wherein the antigen binding protein binds to a polypeptide of

SEQ ID NO: 18 TtSlyDcys-Her3,

-   -   and

-   b) wherein the antigen binding protein binds to a polypeptide of

SEQ ID NO: 22 TtSlyDcys-Her4.

The invention further provides an isolated antigen binding protein thatbinds to human HER3 and that binds to human HER4,

-   a) wherein the antigen binding protein binds within an amino acid    sequence of PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1) which is comprised in    a polypeptide of SEQ ID NO: 18 (TtSlyDcas-Her3), and-   b) wherein the antigen binding protein binds within an amino acid    sequence of PQTFVYNPTTFQLEHNFNA (SEQ ID NO:2) which is comprised in    a polypeptide of SEQ ID NO: 22 (TtSlyDcas-Her4).

The invention provides an isolated antibody that binds to human HER3 andthat binds to human HER4,

-   a) wherein the antibody binds to a polypeptide selected from the    group consisting of:

SEQ ID NO: 13 TtSlyD-FKBP-Her3, SEQ ID NO: 17 TtSlyDcas-Her3, SEQ ID NO:18 TtSlyDcys-Her3, SEQ ID NO: 19 TgSlyDser-Her3, and SEQ ID NO: 20TgSlyDcys-Her3,

-   -   and

-   b) wherein the antibody binds to a polypeptide selected from the    group consisting of:

SEQ ID NO: 21 TtSlyDcas-Her4, SEQ ID NO: 22 TtSlyDcys-Her4, SEQ ID NO:23 TgSlyDser-Her4, and SEQ ID NO: 24 TgSlyDcys-Her4.

The invention further provides an isolated antibody that binds to humanHER3 and that binds to human HER4,

-   a) wherein the antibody binds within an amino acid sequence of    PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1) which is comprised in a    polypeptide selected from the group consisting of:

SEQ ID NO: 13 TtSlyD-FKBP-Her3, SEQ ID NO: 17 TtSlyDcas-Her3, SEQ ID NO:18 TtSlyDcys-Her3, SEQ ID NO: 19 TgSlyDser-Her3, and SEQ ID NO: 20TgSlyDcys-Her3;

-   -   and

-   b) wherein the antibody binds within an amino acid sequence of    PQTFVYNPTTFQLEHNFNA (SEQ ID NO:2) which is comprised in a    polypeptide selected from the group consisting of:

SEQ ID NO: 21 TtSlyDcas-Her4, SEQ ID NO: 22 TtSlyDcys-Her4, SEQ ID NO:23 TgSlyDser-Her4, and SEQ ID NO: 24 TgSlyDcys-Her4.

The invention provides an isolated antibody that binds to human HER3 andthat binds to human HER4,

-   a) wherein the antibody binds to a polypeptide of

SEQ ID NO: 18 TtSlyDcys-Her3,

-   -   and

-   b) wherein the antibody binds to a polypeptide of

SEQ ID NO: 22 TtSlyDcys-Her4.

The invention further provides an isolated antibody that binds to humanHER3 and that binds to human HER4,

-   a) wherein the antibody binds within an amino acid sequence of    PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1) which is comprised in a    polypeptide of SEQ ID NO: 18 (TtSlyDcas-Her3), and-   b) wherein the antibody binds within an amino acid sequence of    PQTFVYNPTTFQLEHNFNA (SEQ ID NO:2) which is comprised in a    polypeptide of SEQ ID NO: 22 (TtSlyDcas-Her4).

In one aspect, The invention provides an isolated antibody that binds tohuman HER3 and binds to human HER4, wherein the antibody binds within anamino acid sequence of PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1) of human HER3and binds within an amino acid sequence of PQTFVYNPTTFQLEHNFNA (SEQ IDNO:2) of human HER4.

In certain embodiments, the invention provides an isolated antibody thatbinds to human HER3 and that binds to human HER4, wherein the antibodyhas one or more of the following properties (also each combination ofeach single property is contemplated herein):

-   a) the antibody binds to the amino acid sequence of SEQ ID NO:1;    and/or-   b) the antibody binds to the amino acid sequence SEQ ID NO:1 in    activated HER3; and/or-   c) the antibody binds within an amino acid sequence of    PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1) which is comprised in a    polypeptide selected from the group consisting of:

SEQ ID NO: 13 TtSlyD-FKBP-Her3, SEQ ID NO: 17 TtSlyDcas-Her3, SEQ ID NO:18 TtSlyDcys-Her3, SEQ ID NO: 19 TgSlyDser-Her3, and SEQ ID NO: 20TgSlyDcys-Her3;

-   -   and/or

-   d) the antibody binds to a polypeptide selected from the group    consisting of:

SEQ ID NO: 13 TtSlyD-FKBP-Her3, SEQ ID NO: 17 TtSlyDcas-Her3, SEQ ID NO:18 TtSlyDcys-Her3, SEQ ID NO: 19 TgSlyDser-Her3, and SEQ ID NO: 20TgSlyDcys-Her3;

-   -   and/or

-   e) the antibody binds to the β-hairpin region of HER3; and/or

-   f) the antibody inhibits the heterodimerisation of HER3/HER2    heterodimers in MCF-7 cells in a HER3/HER2 coprecipitation assay    (see Example 7); and/or

-   g) the antibody binds to HER3-ECD with a ratio of the association    constant (Ka) in presence of Heregulin (Ka (+Heregulin)) and in    absence of Heregulin (Ka (−Heregulin)) of 4.0 or higher (Ka    (+Heregulin))/(Ka (−Heregulin)) (see Example 3b); and/or

-   h) the antibody binds to HER3-ECD with a ratio of the Molar Ratio MR    of binding in presence of Heregulin (MR (+Heregulin)) and in absence    of Heregulin (MR (−Heregulin)) of 2.0 or higher (MR    (+Heregulin))/(MR (−Heregulin)) (see Example 3b); and/or

-   i) the antibody binds to the amino acid sequence of SEQ ID NO:2;    and/or

-   j) the antibody binds to the amino acid sequence SEQ ID NO:2 in    activated HER4; and/or

-   k) the antibody binds within an amino acid sequence of    PQTFVYNPTTFQLEHNFNA (SEQ ID NO:2) which is comprised in a    polypeptide selected from the group consisting of:

SEQ ID NO: 21 TtSlyDcas-Her4, SEQ ID NO: 22 TtSlyDcys-Her4, SEQ ID NO:23 TgSlyDser-Her4, and SEQ ID NO: 24 TgSlyDcys-Her4;

-   -   and/or

-   l) the antibody binds to a polypeptide selected from the group    consisting of:

SEQ ID NO: 21 TtSlyDcas-Her4, SEQ ID NO: 22 TtSlyDcys-Her4, SEQ ID NO:23 TgSlyDser-Her4, and SEQ ID NO: 24 TgSlyDcys-Her4;

-   -   and/or

-   m) the antibody binds to the β-hairpin region of HER4; and/or

-   n) the antibody binds to HER4-ECD with a ratio of the association    constant (Ka) in presence of Heregulin (Ka (+Heregulin)) and in    absence of Heregulin (Ka (−Heregulin)) of 20.0 or higher (Ka    (+Heregulin))/(Ka (−Heregulin)) (see Example 3b); and/or

-   o) the antibody binds to HER4-ECD with a ratio of the Molar Ratio MR    of binding in presence of Heregulin (MR (+Heregulin)) and in absence    of Heregulin (MR (−Heregulin)) of 5.0 or higher (MR    (+Heregulin))/(MR (−Heregulin)) (see Example 3b); and/or

-   p) the antibody shows as monovalent Fab fragment the same or higher    biological activity as compared to its bivalent parent full length    antibody (in a HER3 phosphorylation inhibition assay in MCF-7 cells,    when compared in equimolar amounts) (see Example 6); and/or

-   q) the antibody inhibits the HER3 phosporylation in MCF-7 cells    (with at least 90% at a concentration of 6.66 nM) (see Example 6);    and/or

-   r) the antibody does not compete for binding to HER3 with    Heregulin/induces binding of Heregulin to HER3 (see Example 5);    and/or

-   s) the antibody inhibits the proliferation of MDA-MB-175 tumor cells    (with an EC50 of 5 μg/ml or lower); and/or

-   t) the antibody shows tumor growth inhibitory activity in vivo;    and/or

-   u) the antibody binds with an affinity of a KD value ≤1×10⁻⁸ M to    HER3-ECD (in one embodiment with a KD value of 1×10⁻⁸ M to 1×10⁻¹³    M; (in one embodiment with a KD value of 1×10⁻⁹ M to 1×10⁻¹³ M);    and/or

-   v) the antibody binds with an affinity of a KD value ≤1×10⁻⁸ M to    HER4-ECD (in one embodiment with a KD value of 1×10⁻⁸ M to 1×10⁻¹³    M; (in one embodiment with a KD value of 1×10⁻⁹ M to 1×10⁻¹³ M);    and/or

-   w) the antibody binds to a polypeptide consisting of VYNKLTFQLEP    (SEQ ID NO:43) and to a polypeptide of consisting of VYNPTTFQLE (SEQ    ID NO:44); and/or

-   x) the antibody binds to a polypeptide consisting of VYNKLTFQLEP    (SEQ ID NO:43); and/or

-   y) the antibody binds to a polypeptide consisting of VYNPTTFQLE (SEQ    ID NO:44); and/or

-   z) the antibody binds in a FACS assay to HER3 expressing T47D cells;    and wherein the antibody induces an at least 25% higher percentage    of internalization of HER3 in the presence of Heregulin as compared    to the percentage of internalization of HER3 in the presence of    Heregulin when measured after 1 h after antibody exposure. (see    example 2e).

In certain embodiments, the invention provides an isolated antibody thatbinds to human HER3 and that binds to human HER4, wherein the antibodyhas one or more of the following properties (also each combination ofeach single property is contemplated herein):

-   -   a) the antibody binds to the amino acid sequence of SEQ ID NO:1;        and the antibody binds to the amino acid sequence of SEQ ID        NO:2;    -   and/or    -   b) the antibody binds to the amino acid sequence SEQ ID NO:1 in        activated HER3; and    -   the antibody binds to the amino acid sequence SEQ ID NO:2 in        activated HER4;    -   and/or    -   c) the antibody binds within an amino acid sequence of        PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1) which is comprised in a        polypeptide selected from the group consisting of:

SEQ ID NO: 13 TtSlyD-FKBP-Her3, SEQ ID NO: 17 TtSlyDcas-Her3, SEQ ID NO:18 TtSlyDcys-Her3, SEQ ID NO: 19 TgSlyDser-Her3, and SEQ ID NO: 20TgSlyDcys-Her3;

-   -   and the antibody binds within an amino acid sequence of        PQTFVYNPTTFQLEHNFNA (SEQ ID NO:2) which is comprised in a        polypeptide selected from the group consisting of:

SEQ ID NO: 21 TtSlyDcas-Her4, SEQ ID NO: 22 TtSlyDcys-Her4, SEQ ID NO:23 TgSlyDser-Her4, and SEQ ID NO: 24 TgSlyDcys-Her4;

-   -   and/or    -   d) the antibody binds to a polypeptide selected from the group        consisting of:

SEQ ID NO: 13 TtSlyD-FKBP-Her3, SEQ ID NO: 17 TtSlyDcas-Her3, SEQ ID NO:18 TtSlyDcys-Her3, SEQ ID NO: 19 TgSlyDser-Her3, and SEQ ID NO: 20TgSlyDcys-Her3;

-   -   the antibody binds to a polypeptide selected from the group        consisting of:

SEQ ID NO: 21 TtSlyDcas-Her4, SEQ ID NO: 22 TtSlyDcys-Her4, SEQ ID NO:23 TgSlyDser-Her4, and SEQ ID NO: 24 TgSlyDcys-Her4;

-   -   and/or    -   e) the antibody binds to the β-hairpin region of HER3; and the        antibody binds to the β-hairpin region of HER4;    -   and/or    -   f) the antibody binds to HER3-ECD with a ratio of the        association constant (Ka) in presence of Heregulin (Ka        (+Heregulin)) and in absence of Heregulin (Ka (−Heregulin)) of        4.0 or higher (Ka (+Heregulin))/(Ka (−Heregulin)) (see Example        3b); and    -   the antibody binds to HER4-ECD with a ratio of the association        constant (Ka) in presence of Heregulin (Ka (+Heregulin)) and in        absence of Heregulin (Ka (−Heregulin)) of 20.0 or higher (Ka        (+Heregulin))/(Ka (−Heregulin)) (see Example 3b);    -   and/or    -   g) the antibody binds to HER3-ECD with a ratio of the Molar        Ratio MR of binding in presence of Heregulin (MR (+Heregulin))        and in absence of Heregulin (MR (−Heregulin)) of 2.0 or higher        (MR (+Heregulin))/(MR (−Heregulin)) (see Example 3b); and    -   the antibody binds to HER4-ECD with a ratio of the Molar Ratio        MR of binding in presence of Heregulin (MR (+Heregulin)) and in        absence of Heregulin (MR (−Heregulin)) of 5.0 or higher (MR        (+Heregulin))/(MR (−Heregulin)) (see Example 3b);    -   and/or    -   h) the antibody binds with an affinity of a KD value ≤1×10⁻⁸ M        to HER3-ECD (in one embodiment with a KD value of 1×10⁻⁸ M to        1×10⁻¹³ M; (in one embodiment with a KD value of 1×10⁻⁹ M to        1×10⁻¹³ M); and    -   the antibody binds with an affinity of a KD value ≤1×10⁻⁸ M to        HER4-ECD (in one embodiment with a KD value of 1×10⁻⁸ M to        1×10⁻¹³ M; (in one embodiment with a KD value of 1×10⁻⁹ M to        1×10⁻¹³ M);    -   and/or    -   i) the antibody binds to a polypeptide with a length of 15 amino        acids comprising the amino acid sequence VYNKLTFQLEP (SEQ ID        NO:43) and to a polypeptide with a length of 15 amino acids        comprising the amino acid sequence VYNPTTFQLE (SEQ ID NO:44);        and/or    -   j) the antibody binds to a polypeptide with a length of 15 amino        acids comprising the amino acid sequence VYNKLTFQLEP (SEQ ID        NO:43); and/or    -   k) the antibody binds to a polypeptide with a length of 15 amino        acids comprising the amino acid sequence VYNPTTFQLE (SEQ ID        NO:44).

In certain embodiments, the invention provides an isolated antibody thatbinds to human HER3 and that binds to human HER4, wherein:

-   -   the antibody binds to the amino acid sequence of SEQ ID NO:1;        and    -   the antibody binds to the amino acid sequence of SEQ ID NO:2.

In certain embodiments, the invention provides an isolated antibody thatbinds to human HER3 and that binds to human HER4, wherein:

-   -   the antibody binds to the amino acid sequence SEQ ID NO:1 in        activated HER3; and    -   the antibody binds to the amino acid sequence SEQ ID NO:2 in        activated HER4;

In certain embodiments, the invention provides an isolated antibody thatbinds to human HER3 and that binds human HER4, wherein the antibodybinds to a polypeptide with a length of 15 amino acids comprising theamino acid sequence VYNKLTFQLEP (SEQ ID NO:43) (of human HER3) and to apolypeptide with a length of 15 amino acids comprising the amino acidsequence VYNPTTFQLE (SEQ ID NO:44) (of human HER4).

In one aspect, the invention provides an anti-HER3/HER4 antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:25; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:26; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:27; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:28; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:29; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:30.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:25; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:26; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:27. In one embodiment,the antibody comprises HVR-H3 comprising the amino acid sequence of SEQID NO:27. In another embodiment, the antibody comprises HVR-H3comprising the amino acid sequence of SEQ ID NO:27 and HVR-L3 comprisingthe amino acid sequence of SEQ ID NO:30. In a further embodiment, theantibody comprises HVR-H3 comprising the amino acid sequence of SEQ IDNO:27, HVR-L3 comprising the amino acid sequence of SEQ ID NO:30, andHVR-H1 comprising the amino acid sequence of SEQ ID NO:25. In a furtherembodiment, the antibody comprises (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO:25; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO:26; and (c) HVR-H3 comprising the amino acid sequence ofSEQ ID NO:27.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:28; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:29; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:30. In one embodiment,the antibody comprises (a) HVR-L1 comprising the amino acid sequence ofSEQ ID NO:28; (b) HVR-L2 comprising the amino acid sequence of SEQ IDNO:29; and (c) HVR-L3 comprising the amino acid sequence of SEQ IDNO:30.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:25, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:26, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO:27; and (b) a VL domain comprising at least one, at least two,or all three VL HVR sequences selected from (i) HVR-L1 comprising theamino acid sequence of SEQ ID NO:28, (ii) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:29, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:30.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:25; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:26; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:27; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:28; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:29; and (f) HVR-L3comprising an amino acid sequence selected from SEQ ID NO:30.

In one aspect, the invention provides an anti-HER3/HER4 antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:38; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:39; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:27; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:40; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:41; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:30.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:38; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:39; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:27. In one embodiment,the antibody comprises HVR-H3 comprising the amino acid sequence of SEQID NO:27. In another embodiment, the antibody comprises HVR-H3comprising the amino acid sequence of SEQ ID NO:27 and HVR-L3 comprisingthe amino acid sequence of SEQ ID NO:30. In a further embodiment, theantibody comprises HVR-H3 comprising the amino acid sequence of SEQ IDNO:27, HVR-L3 comprising the amino acid sequence of SEQ ID NO:30, andHVR-H2 comprising the amino acid sequence of SEQ ID NO:39. In a furtherembodiment, the antibody comprises (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO:38; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO:39; and (c) HVR-H3 comprising the amino acid sequence ofSEQ ID NO:27.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:40; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:41; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:30. In one embodiment,the antibody comprises (a) HVR-L1 comprising the amino acid sequence ofSEQ ID NO:40; (b) HVR-L2 comprising the amino acid sequence of SEQ IDNO:41; and (c) HVR-L3 comprising the amino acid sequence of SEQ IDNO:30.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:38, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:39, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO:27; and (b) a VL domain comprising at least one, at least two,or all three VL HVR sequences selected from (i) HVR-L1 comprising theamino acid sequence of SEQ ID NO:40, (ii) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:41, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:30.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:38; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:39; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:27; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:40; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:41; and (f) HVR-L3comprising an amino acid sequence selected from SEQ ID NO:30.

In one aspect, the invention provides an anti-HER3/HER4 antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:38; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:39; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:27; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:40; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:42; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:30.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:38; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:39; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:27. In one embodiment,the antibody comprises HVR-H3 comprising the amino acid sequence of SEQID NO:27. In another embodiment, the antibody comprises HVR-H3comprising the amino acid sequence of SEQ ID NO:27 and HVR-L3 comprisingthe amino acid sequence of SEQ ID NO:30. In a further embodiment, theantibody comprises HVR-H3 comprising the amino acid sequence of SEQ IDNO:27, HVR-L3 comprising the amino acid sequence of SEQ ID NO:30, andHVR-H2 comprising the amino acid sequence of SEQ ID NO:39. In a furtherembodiment, the antibody comprises (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO:38; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO:39; and (c) HVR-H3 comprising the amino acid sequence ofSEQ ID NO:27.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:40; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:42; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:30. In one embodiment,the antibody comprises (a) HVR-L1 comprising the amino acid sequence ofSEQ ID NO:40; (b) HVR-L2 comprising the amino acid sequence of SEQ IDNO:42; and (c) HVR-L3 comprising the amino acid sequence of SEQ IDNO:30.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:38, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:39, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO:27; and (b) a VL domain comprising at least one, at least two,or all three VL HVR sequences selected from (i) HVR-L1 comprising theamino acid sequence of SEQ ID NO:40, (ii) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:42, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:30.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:38; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:39; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:27; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:40; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:42; and (f) HVR-L3comprising an amino acid sequence selected from SEQ ID NO:30.

In one aspect, the invention provides an anti-HER3/HER4 antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:25; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:39; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:27; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:40; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:41; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:30.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:25; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:39; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:27. In one embodiment,the antibody comprises HVR-H3 comprising the amino acid sequence of SEQID NO:27. In another embodiment, the antibody comprises HVR-H3comprising the amino acid sequence of SEQ ID NO:27 and HVR-L3 comprisingthe amino acid sequence of SEQ ID NO:30. In a further embodiment, theantibody comprises HVR-H3 comprising the amino acid sequence of SEQ IDNO:27, HVR-L3 comprising the amino acid sequence of SEQ ID NO:30, andHVR-H2 comprising the amino acid sequence of SEQ ID NO:39. In a furtherembodiment, the antibody comprises (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO:25; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO:39; and (c) HVR-H3 comprising the amino acid sequence ofSEQ ID NO:27.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:40; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:41; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:30. In one embodiment,the antibody comprises (a) HVR-L1 comprising the amino acid sequence ofSEQ ID NO:40; (b) HVR-L2 comprising the amino acid sequence of SEQ IDNO:41; and (c) HVR-L3 comprising the amino acid sequence of SEQ IDNO:30.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:25, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:39, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO:27; and (b) a VL domain comprising at least one, at least two,or all three VL HVR sequences selected from (i) HVR-L1 comprising theamino acid sequence of SEQ ID NO:40, (ii) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:41, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:30.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:25; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:39; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:27; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:40; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:41; and (f) HVR-L3comprising an amino acid sequence selected from SEQ ID NO:30.

In one aspect, the invention provides an anti-HER3/HER4 antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:25; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:39; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:27; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:40; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:42; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:30.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:25; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:39; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:27. In one embodiment,the antibody comprises HVR-H3 comprising the amino acid sequence of SEQID NO:27. In another embodiment, the antibody comprises HVR-H3comprising the amino acid sequence of SEQ ID NO:27 and HVR-L3 comprisingthe amino acid sequence of SEQ ID NO:30. In a further embodiment, theantibody comprises HVR-H3 comprising the amino acid sequence of SEQ IDNO:27, HVR-L3 comprising the amino acid sequence of SEQ ID NO:30, andHVR-H2 comprising the amino acid sequence of SEQ ID NO:39. In a furtherembodiment, the antibody comprises (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO:25; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO:39; and (c) HVR-H3 comprising the amino acid sequence ofSEQ ID NO:27.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:40; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:42; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:30. In one embodiment,the antibody comprises (a) HVR-L1 comprising the amino acid sequence ofSEQ ID NO:40; (b) HVR-L2 comprising the amino acid sequence of SEQ IDNO:42; and (c) HVR-L3 comprising the amino acid sequence of SEQ IDNO:30.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:25, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:39, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO:27; and (b) a VL domain comprising at least one, at least two,or all three VL HVR sequences selected from (i) HVR-L1 comprising theamino acid sequence of SEQ ID NO:40, (ii) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:42, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:30.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:25; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:39; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:27; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:40; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:42; and (f) HVR-L3comprising an amino acid sequence selected from SEQ ID NO:30.

In another aspect, the invention provides an anti-HER3/HER4 antibodycomprising

-   -   i) (a) HVR-H1 comprising the amino acid sequence of SEQ ID        NO:25;    -   (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:26;    -   (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:27;    -   (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:28;    -   (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29;    -   and (f) HVR-L3 comprising the amino acid sequence of SEQ ID        NO:30;    -   ii) or a humanized variant of the HVRs of the antibody under i)        (a), (b), (d) and/or (e).

In another aspect, the invention provides an anti-HER3/HER4 antibodycomprising

-   -   (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:38;    -   (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:39;    -   (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:27;    -   (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:40;    -   (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:41;        and    -   (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:30:

In another aspect, the invention provides an anti-HER3/HER4 antibodycomprising

-   -   (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:38;    -   (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:39;    -   (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:27;    -   (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:40;    -   (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:42;        and    -   (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:30.

In another aspect, the invention provides an anti-HER3/HER4 antibodycomprising

-   -   (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:25;    -   (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:39;    -   (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:27;    -   (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:40;    -   (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:41;        and    -   (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:30.

In another aspect, the invention provides an anti-HER3/HER4 antibodycomprising

-   -   (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:25;    -   (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:39;    -   (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:27;    -   (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:40;    -   (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:42;        and    -   (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:30.

In any of the above embodiments, an anti-HER3/HER4 antibody ishumanized. In one embodiment, an anti-HER3/HER4 antibody comprises HVRsas in any of the above embodiments, and further comprises an acceptorhuman framework, e.g. a human immunoglobulin framework or a humanconsensus framework. In another embodiment, an anti-HER3/HER4 antibodycomprises HVRs as in any of the above embodiments, and further comprisesa VH comprising a framework region of human germline IGHV1-46-01 orIMGT_hVH_1_46 and a VL comprising a framework region of human germlineIGKV3-11-01 or IMGT_hVK_1_39. Frameork regions and sequences of humangermlines are described in Poul, M-A. and Lefranc, M-P., in “Ingénieriedes anticorps banques combinatores” ed. by Lefranc, M-P. and Lefranc,G., Les Editions INSERM, 1997. Human heavy and light chain variableframework regions of all human germlines are listed e.g. in Lefranc, M.P., Current Protocols in Immunology (2000)—Appendix 1P A.1P.1-A.1P.37and are accessible via IMGT, the international ImMunoGeneTicsinformation system® (http://imgt.cines.fr) or viahttp://vbase.mrc-cpe.cam.ac.uk.

In another aspect, an anti-HER3/HER4 antibody comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO:33. In certain embodiments, a VH sequence havingat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identitycontains substitutions (e.g., conservative substitutions), insertions,or deletions relative to the reference sequence, but an anti-HER3/HER4antibody comprising that sequence retains the ability to bind to HER3,and HER4, respectively. In certain embodiments, a total of 1 to 10 aminoacids have been substituted, inserted and/or deleted in SEQ ID NO:33. Incertain embodiments, substitutions, insertions, or deletions occur inregions outside the HVRs (i.e., in the FRs). Optionally, theanti-HER3/HER4 antibody comprises the VH sequence in SEQ ID NO:33,including post-translational modifications of that sequence. In aparticular embodiment, the VH comprises one, two or three HVRs selectedfrom: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:38, (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:39, and (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO:27.

In another aspect, an anti-HER3/HER4 antibody comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO:34. In certain embodiments, a VH sequence havingat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identitycontains substitutions (e.g., conservative substitutions), insertions,or deletions relative to the reference sequence, but an anti-HER3/HER4antibody comprising that sequence retains the ability to bind to HER3,and HER4, respectively. In certain embodiments, a total of 1 to 10 aminoacids have been substituted, inserted and/or deleted in SEQ ID NO:34. Incertain embodiments, substitutions, insertions, or deletions occur inregions outside the HVRs (i.e., in the FRs). Optionally, theanti-HER3/HER4 antibody comprises the VH sequence in SEQ ID NO:34,including post-translational modifications of that sequence. In aparticular embodiment, the VH comprises one, two or three HVRs selectedfrom: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:25, (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:39, and (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO:27.

In another aspect, an anti-HER3/HER4 antibody comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO:35. In certain embodiments, a VH sequence havingat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identitycontains substitutions (e.g., conservative substitutions), insertions,or deletions relative to the reference sequence, but an anti-HER3/HER4antibody comprising that sequence retains the ability to bind to HER3,and HER4, respectively. In certain embodiments, a total of 1 to 10 aminoacids have been substituted, inserted and/or deleted in SEQ ID NO:35. Incertain embodiments, substitutions, insertions, or deletions occur inregions outside the HVRs (i.e., in the FRs). Optionally, theanti-HER3/HER4 antibody comprises the VH sequence in SEQ ID NO:35,including post-translational modifications of that sequence. In aparticular embodiment, the VH comprises one, two or three HVRs selectedfrom: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:25, (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:39, and (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO:27.

In another aspect, an anti-HER3/HER4 antibody is provided, wherein theantibody comprises a light chain variable domain (VL) having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO:36. In certainembodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-HER3/HER4 antibody comprising thatsequence retains the ability to bind to HER3, and HER4, respectively. Incertain embodiments, a total of 1 to 10 amino acids have beensubstituted, inserted and/or deleted in SEQ ID NO:36. In certainembodiments, the substitutions, insertions, or deletions occur inregions outside the HVRs (i.e., in the FRs). Optionally, theanti-HER3/HER4 antibody comprises the VL sequence in SEQ ID NO:36,including post-translational modifications of that sequence. In aparticular embodiment, the VL comprises one, two or three HVRs selectedfrom (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:40; (b)HVR-L2 comprising the amino acid sequence of SEQ ID NO:41; and (c)HVR-L3 comprising the amino acid sequence of SEQ ID NO:30.

In another aspect, an anti-HER3/HER4 antibody is provided, wherein theantibody comprises a light chain variable domain (VL) having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO:37. In certainembodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-HER3/HER4 antibody comprising thatsequence retains the ability to bind to HER3, and HER4, respectively. Incertain embodiments, a total of 1 to 10 amino acids have beensubstituted, inserted and/or deleted in SEQ ID NO:37. In certainembodiments, the substitutions, insertions, or deletions occur inregions outside the HVRs (i.e., in the FRs). Optionally, theanti-HER3/HER4 antibody comprises the VL sequence in SEQ ID NO:37,including post-translational modifications of that sequence. In aparticular embodiment, the VL comprises one, two or three HVRs selectedfrom (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:40; (b)HVR-L2 comprising the amino acid sequence of SEQ ID NO:42; and (c)HVR-L3 comprising the amino acid sequence of SEQ ID NO:30.

In another aspect, an anti-HER3/Her4 antibody is provided, wherein theantibody comprises a VH as in any of the embodiments provided above, anda VL as in any of the embodiments provided above. In one embodiment, theantibody comprises the VH and VL sequences in SEQ ID NO:33 and SEQ IDNO:36, the VH and VL sequences in SEQ ID NO:33 and SEQ ID NO:37 the VHand VL sequences in SEQ ID NO:34 and SEQ ID NO:36, the VH and VLsequences in SEQ ID NO:34 and SEQ ID NO:37, the VH and VL sequences inSEQ ID NO:35 and SEQ ID NO:36, or the VH and VL sequences in SEQ IDNO:33 and SEQ ID NO:37, respectively, including post-translationalmodifications of those sequences.

In another aspect, an anti-HER3/Her4 antibody is provided, wherein theantibody comprises a heavy chain variable domain (VH) sequence having atleast 95%, sequence identity and a light chain variable domain (VL)having at least 95%, or 100% sequence identity to antibody comprising

-   a) the VH and VL sequences in SEQ ID NO:33 and SEQ ID NO:36,-   b) the VH and VL sequences in SEQ ID NO:33 and SEQ ID NO:37-   c) the VH and VL sequences in SEQ ID NO:34 and SEQ ID NO:36,-   d) the VH and VL sequences in SEQ ID NO:34 and SEQ ID NO:37,-   e) the VH and VL sequences in SEQ ID NO:35 and SEQ ID NO:36, or-   f) the VH and VL sequences in SEQ ID NO:33 and SEQ ID NO:37,    respectively,

wherein the antibody has one or more of the following properties:

In certain embodiments, the invention provides an isolated antibody thatbinds to human HER3 and that binds to human HER4, wherein the antibodyhas one or more of the following properties: the antibody

-   a) binds within an amino acid sequence of PQPLVYNKLTFQLEPNPHT (SEQ    ID NO:1) which is comprised in a polypeptide selected from the group    consisting of:

SEQ ID NO: 13 TtSlyD-FKBP-Her3, SEQ ID NO: 17 TtSlyDcas-Her3, SEQ ID NO:18 TtSlyDcys-Her3, SEQ ID NO: 19 TgSlyDser-Her3, and SEQ ID NO: 20TgSlyDcys-Her3;

-   b) binds to a polypeptide selected from the group consisting of:

SEQ ID NO: 13 TtSlyD-FKBP-Her3, SEQ ID NO: 17 TtSlyDcas-Her3, SEQ ID NO:18 TtSlyDcys-Her3, SEQ ID NO: 19 TgSlyDser-Her3, and SEQ ID NO: 20TgSlyDcys-Her3;

-   c) inhibits the heterodimerisation of HER3/HER2 heterodimers in    MCF-7 cells in a HER3/HER2 coprecipitation assay;-   d) binds within an amino acid sequence of PQTFVYNPTTFQLEHNFNA (SEQ    ID NO:2) which is comprised in a polypeptide selected from the group    consisting of:

SEQ ID NO: 21 TtSlyDcas-Her4, SEQ ID NO: 22 TtSlyDcys-Her4, SEQ ID NO:23 TgSlyDser-Her4, and SEQ ID NO: 24 TgSlyDcys-Her4;

-   e) binds to a polypeptide selected from the group consisting of:

SEQ ID NO: 21 TtSlyDcas-Her4, SEQ ID NO: 22 TtSlyDcys-Her4, SEQ ID NO:23 TgSlyDser-Her4, and SEQ ID NO: 24 TgSlyDcys-Her4;

-   f) shows as monovalent Fab fragment the same or higher biological    activity as compared to its bivalent parent full length antibody    (when compared in equimolar amounts in a HER3 phosphorylation    inhibition assay in MCF-7 cells);-   g) shows tumor growth inhibitory activity in vivo;-   h) binds with an affinity of a KD value ≤1×10-8 M to HER3-ECD (in    one embodiment with a KD value of 1×10-8 M to 1×10-13 M; (in one    embodiment with a KD value of 1×10-9 M to 1×10-13 M);-   i) binds with an affinity of a KD value ≤1×10-8 M to HER4-ECD (in    one embodiment with a KD value of 1×10-8 M to 1×10-13 M; (in one    embodiment with a KD value of 1×10-9 M to 1×10-13 M);-   j) wherein the antibody binds to a polypeptide consisting of    VYNKLTFQLEP (SEQ ID NO:43) or to a polypeptide of consisting of    VYNPTTFQLE (SEQ ID NO:44);-   k) wherein the antibody binds to a polypeptide consisting of    VYNKLTFQLEP (SEQ ID NO:43); and/or-   l) wherein the antibody binds to a polypeptide consisting of    VYNPTTFQLE (SEQ ID NO:44).

In a further aspect, the invention provides an antibody that binds tothe same epitope as an anti-HER3/HER4 antibody provided herein. Forexample, in certain embodiments, an antibody is provided that binds tothe same epitope as an anti-HER3/HER4 antibody comprising a VH sequenceof SEQ ID NO:31 and a VL sequence of SEQ ID NO:32. In certainembodiments, an antibody is provided that binds to an epitope within afragment of human HER3 consisting of amino acids VYNKLTFQLEP (SEQ IDNO:43) and/or that binds to an epitope within a fragment of human HER4consisting of amino acids VYNPTTFQLE (SEQ ID NO:44).

1. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant KD of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or≤0.001 nM (e.g. 10⁻⁸M or less, e.g. from 10⁻⁸M to 10⁻¹³M, e.g., from10⁻⁹M to 10⁻¹³M).

In one preferred embodiment, KD is measured using surface plasmonresonance assays using a BIACORE®) at 25° C. with immobilized antigenCMS chips at ˜10 response units (RU). Briefly, carboxymethylated dextranbiosensor chips (CM5, BIACORE, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (˜0.2μM) before injection at a flow rate of 5 μl/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, 1 M ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20(TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate of approximately25 μl/min. Association rates (k_(on) or ka) and dissociation rates(k_(off) or kd) are calculated using a simple one-to-one Langmuirbinding model (BIACORE ® Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant KD is calculated as the ratio kd/ka(k_(off)/k_(on.)) See, e.g., Chen, Y. et al., J. Mol. Biol. 293 (1999)865-881. If the on-rate exceeds 10⁶ M⁻¹ s⁻¹ by the surface plasmonresonance assay above, then the on-rate can be determined by using afluorescent quenching technique that measures the increase or decreasein fluorescence emission intensity (excitation=295 nm; emission=340 nm,16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form)in PBS, pH 7.2, in the presence of increasing concentrations of antigenas measured in a spectrometer, such as a stop-flow equippedspectrophotometer (Aviv Instruments) or a 8000-series SLM-AMINCO™spectrophotometer (ThermoSpectronic) with a stirred cuvette.

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson, P. J. etal., Nat. Med. 9 (2003) 129-134. For a review of scFv fragments, see,e.g., Plueckthun, A., In; The Pharmacology of Monoclonal Antibodies,Vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York (1994),pp. 269-315; see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)₂ fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 0 404 097; WO1993/01161; Hudson, P. J. et al., Nat. Med. 9 (2003) 129-134; andHolliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448.Triabodies and tetrabodies are also described in Hudson, P. J. et al.,Nat. Med. 9 (20039 129-134).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage), asdescribed herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison, S. L. et al., Proc. Natl. Acad. Sci. USA 81(1984) 6851-6855). In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro, J. C. and Fransson, J., Front. Biosci. 13 (2008) 1619-1633, andare further described, e.g., in Riechmann, I. et al., Nature 332 (1988)323-329; Queen, C. et al., Proc. Natl. Acad. Sci. USA 86 (1989)10029-10033; U.S. Pat. Nos. 5, 821,337, 7,527,791, 6,982,321, and7,087,409; Kashmiri, S. V. et al., Methods 36 (2005) 25-34 (describingSDR (a-CDR) grafting); Padlan, E. A., Mol. Immunol. 28 (1991) 489-498(describing “resurfacing”); Dall'Acqua, W. F. et al., Methods 36 (2005)43-60 (describing “FR shuffling”); and Osbourn, J. et al., Methods 36(2005) 61-68 and Klimka, A. et al., Br. J. Cancer 83 (2000) 252-260(describing the “guided selection” approach to FR shuffling). Morea, V.,et al., Methods, Vol 20, Issue 3 (2000) 267-279) and WO2004/006955(approach via canonical structures).

4. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk, M. A. and vande Winkel, J. G., Curr. Opin. Pharmacol. 5 (2001) 368-374 and Lonberg,N., Curr. Opin. Immunol. 20 (2008) 450-459.

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, N., Nat. Biotech. 23 (2005) 1117-1125.See also, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describingXENOMOUSE™ technology; U.S. Pat. No. 5,770,429 describing HuMab®technology; U.S. Pat. No. 7,041,870 describing K-M MOUSE® technology,and U.S. Patent Application Publication No. US 2007/0061900, describingVelociMouse® technology). Human variable regions from intact antibodiesgenerated by such animals may be further modified, e.g., by combiningwith a different human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor, D.,J. Immunol. 133 (1984) 3001-3005; Brodeur, B. R. et al., MonoclonalAntibody Production Techniques and Applications, Marcel Dekker, Inc.,New York (1987), pp. 51-63; and Boerner, P. et al., J. Immunol. 147(1991) 86-95) Human antibodies generated via human B-cell hybridomatechnology are also described in Li, J. et al., Proc. Natl. Acad. Sci.USA 103 (2006) 3557-3562. Additional methods include those described,for example, in U.S. Pat. No. 7,189,826 (describing production ofmonoclonal human IgM antibodies from hybridoma cell lines) and Ni, J.,Xiandai Mianyixue 26 (2006) 265-268 (describing human-human hybridomas).Human hybridoma technology (Trioma technology) is also described inVollmers, H. P. and Brandlein, S., Histology and Histopathology 20(2005) 927-937 and Vollmers, H. P. and Brandlein, S., Methods andFindings in Experimental and Clinical Pharmacology 27 (2005) 185-191.

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

5. Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are reviewed, e.g., inHoogenboom, H. R. et al., Methods in Molecular Biology 178 (2001) 1-37and further described, e.g., in the McCafferty, J. et al., Nature 348(1990) 552-554; Clackson, T. et al., Nature 352 (1991) 624-628; Marks,J. D. et al., J. Mol. Biol. 222 (1992) 581-597; Marks, J. D. andBradbury, A., Methods in Molecular Biology 248 (2003) 161-175; Sidhu, S.S. et al., J. Mol. Biol. 338 (2004) 299-310; Lee, C. V. et al., J. Mol.Biol. 340 (2004) 1073-1093; Fellouse, F. A., Proc. Natl. Acad. Sci. USA101 (2004) 12467-12472; and Lee, C. V. et al., J. Immunol. Methods 284(2004) 119-132.

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter, G. et al., Ann. Rev.Immunol. 12 (1994) 433-455. Phage typically display antibody fragments,either as single-chain Fv (scFv) fragments or as Fab fragments.Libraries from immunized sources provide high-affinity antibodies to theimmunogen without the requirement of constructing hybridomas.Alternatively, the naive repertoire can be cloned (e.g., from human) toprovide a single source of antibodies to a wide range of non-self andalso self antigens without any immunization as described by Griffiths,A. D. et al., EMBO J. 12 (1993) 725-734. Finally, naive libraries canalso be made synthetically by cloning non-rearranged V-gene segmentsfrom stem cells, and using PCR primers containing random sequence toencode the highly variable CDR3 regions and to accomplish rearrangementin vitro, as described by Hoogenboom, H. R. and Winter, G., J. Mol.Biol. 227 (1992) 381-388. Patent publications describing human antibodyphage libraries include, for example: U.S. Pat. No. 5,750,373, and USPatent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000,2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

6. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, e.g. a bispecific antibody. Multispecific antibodies aremonoclonal antibodies that have binding specificities for at least twodifferent sites. In certain embodiments, one of the bindingspecificities is for HER3/HER4 and the other is for any other antigen.Bispecific antibodies may also be used to localize cytotoxic agents tocells which express HER3 or HER4. Bispecific antibodies can be preparedas full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein, C.and Cuello, A. C., Nature 305 (1983) 537-540, WO 93/08829, andTraunecker, A. et al., EMBO J. 10 (1991) 3655-3659), and “knob-in-hole”engineering (see, e.g., U.S. Pat. No. 5,731,168). Multi-specificantibodies may also be made by engineering electrostatic steeringeffects for making antibody Fc-heterodimeric molecules (WO 2009/089004);cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat.No. 4,676,980, and Brennan, M. et al., Science 229 (1985) 81-83); usingleucine zippers to produce bi-specific antibodies (see, e.g., Kostelny,S. A. et al., J. Immunol. 148 (1992) 1547-1553; using “diabody”technology for making bispecific antibody fragments (see, e.g.,Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448);and using single-chain Fv (sFv) dimers (see, e.g. Gruber, M et al., J.Immunol. 152 (1994) 5368-5374); and preparing trispecific antibodies asdescribed, e.g., in Tutt, A. et al., J. Immunol. 147 (1991) 60-69).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576).

The antibody or fragment herein also includes a “Dual Acting Fab” or“DAF” comprising an antigen binding site that binds to HER3/HER4 as wellas another, different antigen (see, US 2008/0069820, for example).

The antibody or fragment herein also includes multispecific antibodiesdescribed in

WO 2009/080251, WO 2009/080252, WO 2009/080253, WO 2009/080254, WO2010/112193, WO 2010/115589, WO 2010/136172, WO 2010/145792, and WO2010/145793.

7. Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodiesprovided herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in Table 1 under the heading of “preferred substitutions”. Moresubstantial changes are provided in Table 1 under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Leu Norleucine Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g. bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, P. S.,Methods Mol. Biol. 207 (2008) 179-196), and/or SDRs (a-CDRs), with theresulting variant VH or VL being tested for binding affinity. Affinitymaturation by constructing and reselecting from secondary libraries hasbeen described, e.g., in Hoogenboom, H. R. et al. in Methods inMolecular Biology 178 (2002) 1-37. In some embodiments of affinitymaturation, diversity is introduced into the variable genes chosen formaturation by any of a variety of methods (e.g., error-prone PCR, chainshuffling, or oligonucleotide-directed mutagenesis). A secondary libraryis then created. The library is then screened to identify any antibodyvariants with the desired affinity. Another method to introducediversity involves HVR-directed approaches, in which several HVRresidues (e.g., 4-6 residues at a time) are randomized. HVR residuesinvolved in antigen binding may be specifically identified, e.g., usingalanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 inparticular are often targeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may be outside of HVR “hotspots” orSDRs. In certain embodiments of the variant VH and VL sequences providedabove, each HVR either is unaltered, or contains no more than one, twoor three amino acid substitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham, B. C. and Wells, J. A., Science244 (1989) 1081-1085. In this method, a residue or group of targetresidues (e.g., charged residues such as arg, asp, his, lys, and glu)are identified and replaced by a neutral or negatively charged aminoacid (e.g., alanine or polyalanine) to determine whether the interactionof the antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex toidentify contact points between the antibody and antigen. Such contactresidues and neighboring residues may be targeted or eliminated ascandidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

b) Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright, A. and Morrison, S. L., TIBTECH 15 (1997)26-32. The oligosaccharide may include various carbohydrates, e.g.,mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, aswell as a fucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. For example, the amount of fucose in such antibody may be from1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amountof fucose is determined by calculating the average amount of fucosewithin the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn 297 (e. g. complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (Eunumbering of Fc region residues); however, Asn297 may also be locatedabout ±3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, e.g., US 2003/0157108; US 2004/0093621. Examples of publicationsrelated to “defucosylated” or “fucose-deficient” antibody variantsinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO 2005/053742; WO 2002/031140; Okazaki, A.et al., J. Mol. Biol. 336 (2004) 1239-1249; Yamane-Ohnuki, N. et al.,Biotech. Bioeng. 87 (2004) 614-622. Examples of cell lines capable ofproducing defucosylated antibodies include Lec13 CHO cells deficient inprotein fucosylation (Ripka, J. et al., Arch. Biochem. Biophys. 249(1986) 533-545; US 2003/0157108; and WO 2004/056312, especially atExample 11), and knockout cell lines, such asalpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g.,Yamane-Ohnuki, N. et al., Biotech. Bioeng. 87 (2004) 614-622; Kanda, Y.et al., Biotechnol. Bioeng. 94 (2006) 680-688; and WO 2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878; U.S. Pat. No.6,602,684; and US 2005/0123546. Antibody variants with at least onegalactose residue in the oligosaccharide attached to the Fc region arealso provided. Such antibody variants may have improved CDC function.Such antibody variants are described, e.g., in WO 1997/30087; WO1998/58964; and WO 1999/22764.

c) Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express Fc(RIII only, whereas monocytes express FcgammaRI,FcgammaRII and FcgammaRIII FcR expression on hematopoietic cells issummarized in Table 3 on page 464 of Ravetch, J. V. and Kinet, J. P.,Annu. Rev. Immunol. 9 (1991) 457-492. Non-limiting examples of in vitroassays to assess ADCC activity of a molecule of interest is described inU.S. Pat. No. 5,500,362 (see, e.g. Hellstrom, I. et al., Proc. Natl.Acad. Sci. USA 83 (1986) 7059-7063; and Hellstrom, I. et al., Proc.Natl. Acad. Sci. USA 82 (1985) 1499-1502); U.S. Pat. No. 5,821,337 (seeBruggemann, M. et al., J. Exp. Med. 166 (1987) 1351-1361).Alternatively, non-radioactive assays methods may be employed (see, forexample, ACTI™ non-radioactive cytotoxicity assay for flow cytometry(CellTechnology, Inc. Mountain View, Calif.; and CytoTox96®non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in an animal model such as that disclosed in Clynes, R.et al., Proc. Natl. Acad. Sci. USA 95 (1998) 652-656. C1q binding assaysmay also be carried out to confirm that the antibody is unable to bindC1q and hence lacks CDC activity. See, e.g., C1q and C3c binding ELISAin WO 2006/029879 and WO 2005/100402. To assess complement activation, aCDC assay may be performed (see, for example, Gazzano-Santoro, H. etal., J. Immunol. Methods 202 (1996) 163-171; Cragg, M. S. et al., Blood101 (2003) 1045-1052; and Cragg, M. S. and M. J. Glennie, Blood 103(2004) 2738-2743). FcRn binding and in vivo clearance/half lifedeterminations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al., Int. Immunol. 18 (2006: 1759-1769).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields, R. L. et al., J. Biol. Chem. 276 (2001) 6591-6604)

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie, E. E. et al., J. Immunol. 164(2000) 4178-4184.

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer, R. L. et al., J. Immunol. 117 (1976)587-593, and Kim, J. K. et al., J. Immunol. 24 (1994) 2429-2434), aredescribed in US 2005/0014934. Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

See also Duncan, A. R. and Winter, G., Nature 322 (1988) 738-740; U.S.Pat. Nos. 5,648,260; 5,624,821; and WO 94/29351 concerning otherexamples of Fc region variants.

d) Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and S400 (EU numbering) of the heavy chain Fc region. Cysteineengineered antibodies may be generated as described, e.g., in U.S. Pat.No. 7,521,541.

e) Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional non-proteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer isattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

In another embodiment, conjugates of an antibody and non-proteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the non-proteinaceous moiety is a carbonnanotube (Kam, N. W. et al., Proc. Natl. Acad. Sci. USA 102 (2005)11600-11605). The radiation may be of any wavelength, and includes, butis not limited to, wavelengths that do not harm ordinary cells, butwhich heat the non-proteinaceous moiety to a temperature at which cellsproximal to the antibody-non-proteinaceous moiety are killed.

B. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid encoding an anti-HER3/HER4 antibody describedherein is provided. Such nucleic acid may encode an amino acid sequencecomprising the VL and/or an amino acid sequence comprising the VH of theantibody (e.g., the light and/or heavy chains of the antibody). In afurther embodiment, one or more vectors (e.g., expression vectors)comprising such nucleic acid are provided. In a further embodiment, ahost cell comprising such nucleic acid is provided. In one suchembodiment, a host cell comprises (e.g., has been transformed with): (1)a vector comprising a nucleic acid that encodes an amino acid sequencecomprising the VL of the antibody and an amino acid sequence comprisingthe VH of the antibody, or (2) a first vector comprising a nucleic acidthat encodes an amino acid sequence comprising the VL of the antibodyand a second vector comprising a nucleic acid that encodes an amino acidsequence comprising the VH of the antibody. In one embodiment, the hostcell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoidcell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of makingan anti-HER3/HER4 antibody is provided, wherein the method comprisesculturing a host cell comprising a nucleic acid encoding the antibody,as provided above, under conditions suitable for expression of theantibody, and optionally recovering the antibody from the host cell (orhost cell culture medium).

For recombinant production of an anti-HER3/HER4 antibody, nucleic acidencoding an antibody, e.g., as described above, is isolated and insertedinto one or more vectors for further cloning and/or expression in a hostcell. Such nucleic acid may be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, K. A., In:Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press,Totowa, N.J. (2003), pp. 245-254, describing expression of antibodyfragments in E. coli.) After expression, the antibody may be isolatedfrom the bacterial cell paste in a soluble fraction and can be furtherpurified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, T. U., Nat. Biotech. 22 (2004) 1409-1414; andLi, H. et al., Nat. Biotech. 24 (2006) 210-215.

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham, F. L. et al., J. Gen Virol. 36(1977) 59-74); baby hamster kidney cells (BHK); mouse sertoli cells (TM4cells as described, e.g., in Mather, J. P., Biol. Reprod. 23 (1980)243-252); monkey kidney cells (CV1); African green monkey kidney cells(VERO-76); human cervical carcinoma cells (HELA); canine kidney cells(MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); humanliver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, asdescribed, e.g., in Mather, J. P. et al., Annals N.Y. Acad. Sci. 383(1982) 44-68; MRC 5 cells; and FS4 cells. Other useful mammalian hostcell lines include Chinese hamster ovary (CHO) cells, including DHFR⁻CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980)4216-4220); and myeloma cell lines such as Y0, NS0 and Sp2/0. For areview of certain mammalian host cell lines suitable for antibodyproduction, see, e.g., Yazaki, P. and Wu, A. M., Methods in MolecularBiology, Vol. 248, Lo, B. K. C. (ed.), Humana Press, Totowa, N.J.(2004), pp. 255-268.

C. Assays and Antibody Selection Methods

Anti-HER3/HER4 antibodies (or antigen binding proteins) provided hereinmay be identified, screened for, or characterized for theirphysical/chemical properties and/or biological activities by variousassays known in the art.

One aspect of the invention is a method for selecting an antibody (orantigen binding protein) that binds to human HER3 and binds to humanHER4, wherein the antibody (or antigen binding protein) binds within anamino acid sequence of PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1) of human HER3and binds within an amino acid sequence of PQTFVYNPTTFQLEHNFNA (SEQ IDNO:2) of human HER4; wherein

-   a) at least one polypeptide selected from the group consisting of:

SEQ ID NO: 13 TtSlyD-FKBP-Her3, SEQ ID NO: 17 TtSlyDcas-Her3, SEQ ID NO:18 TtSlyDcys-Her3, SEQ ID NO: 19 TgSlyDser-Her3, and SEQ ID NO: 20TgSlyDcys-Her3,which comprises the amino acid sequence of SEQ ID NO:1;

and

-   b) at least one polypeptide selected from the group consisting of:

SEQ ID NO: 21 TtSlyDcas-Her4, SEQ ID NO: 22 TtSlyDcys-Her4, SEQ ID NO:23 TgSlyDser-Her4, and SEQ ID NO: 24 TgSlyDcys-Her4,

which comprises the amino acid sequence of SEQ ID NO:2;

are used (in a binding assay) to select antibodies (or antigen bindingproteins), which show binding to both, the at least one polypeptideunder a) and the at least one polypeptide under b)

and thereby selecting an antibody (or antigen binding protein) thatbinds within an amino acid sequence of PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1)(within human HER3) and within an amino acid sequence ofPQTFVYNPTTFQLEHNFNA (SEQ ID NO:2) (within human HER4).

In one embodiment the selection method further comprises a step whereinthe selected antibodies (or antigen binding proteins) arecounterscreened with the polypeptides (tested for binding to thepolypeptides) selected from the group consisting of:

SEQ ID NO: 14 TtSlyD-Wildtype SEQ ID NO: 15 TtSlyDcas SEQ ID NO: 16TgSlyDΔIF

to confirm that the selected antibodies (or antigen binding proteins) donot bind to the polypetide scaffolds which are not comprising amino acidsequence of PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1) or the amino acid sequenceof PQTFVYNPTTFQLEHNFNA (SEQ ID NO:2).

The invention provides an antibody (or antigen binding protein) obtainedby such selection method.

A method for selecting an antibody (or antigen binding protein)specifically binding to a human HER3 and human HER4 comprising thefollowing steps:

-   a) determining the binding affinity of a plurality of antibodies (or    antigen binding proteins) to the β-hairpin of HER3 with the amino    acid sequence of SEQ ID NO:1, whereby β-hairpin of HER3 is presented    as polypeptide selected from the group consisting of:

i) SEQ ID NO: 13 TtSlyD-FKBP-Her3, ii) SEQ ID NO: 17 TtSlyDcas-Her3,iii) SEQ ID NO: 18 TtSlyDcys-Her3, iv) SEQ ID NO: 19 TgSlyDser-Her3, andv) SEQ ID NO: 20 TgSlyDcys-Her3,

which comprise the β-hairpin of HER3 with the amino acid sequence of SEQID NO:1,

-   b) selecting the antibody having an apparent complex stability above    a pre-defined threshold level;-   c) determining the binding affinity of the selected antibodies (or    antigen binding proteins) under step b) to the β-hairpin of HER4    with the amino acid sequence of SEQ ID NO:2, whereby β-hairpin of    HER4 is presented as polypeptide selected from the group consisting    of:

i) SEQ ID NO: 21 TtSlyDcas-Her4, ii) SEQ ID NO: 22 TtSlyDcys-Her4, iii)SEQ ID NO: 23 TgSlyDser-Her4, and iv) SEQ ID NO: 24 TgSlyDcys-Her4,

which comprise the β-hairpin of HER4 with the amino acid sequence of SEQID NO:2,

-   d) selecting the antibody (or antigen binding protein) having an    apparent complex stability above a pre-defined threshold level.    1. Binding Assays and Other Assays

In one aspect, an antibody (or antigen binding protein) of the inventionis tested for its antigen binding activity, e.g., by known methods suchas ELISA, Western blot, including surface plasmon resonance (e.g.BIACORE), etc.

In another aspect, competition assays may be used to identify anantibody that competes with M-05-74 for binding to HER3 and/or to HER4.In certain embodiments, such a competing antibody binds to the sameepitope (e.g., a linear or a conformational epitope) that is bound byM-05-74. Detailed exemplary methods for mapping an epitope to which anantibody binds are provided in Morris, G. E. (ed.), Epitope MappingProtocols, In: Methods in Molecular Biology, Vol. 66, Humana Press,Totowa, N.J. (1996). Further methods are described in detail in Example4 using the CelluSpot™ technology.

In an exemplary competition assay, immobilized HER3 or HER4 is incubatedin a solution comprising a first labeled antibody that binds to HER3 orHER4, respectively (e.g., M-05-74) and a second unlabeled antibody thatis being tested for its ability to compete with the first antibody forbinding to HER3 or HER4. The second antibody may be present in ahybridoma supernatant. As a control, immobilized HER3 or HER4 isincubated in a solution comprising the first labeled antibody but notthe second unlabeled antibody. After incubation under conditionspermissive for binding of the first antibody to HER3 or HER4, excessunbound antibody is removed, and the amount of label associated withimmobilized HER3 or HER4 is measured. If the amount of label associatedwith immobilized HER3 or HER4 is substantially reduced in the testsample relative to the control sample, then that indicates that thesecond antibody is competing with the first antibody for binding to HER3or HER4. See Harlow, E. and Lane, D., Antibodies: A Laboratory Manual,Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1988).

2. Activity Assays

In one aspect, assays are provided for identifying anti-HER3/HER4antibodies (or antigen binding proteins) thereof having biologicalactivity. Biological activity may include, e.g., inhibition of HER3 (orHER4) phosphorylation, inhibition of cancer cell proliferation of HER3and/or HER4 expressing or overexpressing cancer cells, inhibition ofHER3/HER2 heterodimerization, (time-dependant) internalization via FACSassay, in vivo tumor growth inhibition in xenograft animal (e.g. mouseor rat) models with xenografted HER3 and/or HER4 expressing oroverexpressing cancer cells. Antibodies having such biological activityin vivo and/or in vitro are also provided.

In certain embodiments, an antibody of the invention is tested for suchbiological activity. Exemplary vitro or in vivo assays for specifiedbiological activities are described in Example 2e, 3, 5 to 9, and 11.

D. Immunoconjugates

The invention also provides immunoconjugates comprising ananti-HER3/HER4 antibody (or antigen binding protein) described hereinconjugated to one or more cytotoxic agents, such as chemotherapeuticagents or drugs, growth inhibitory agents, toxins (e.g., protein toxins,enzymatically active toxins of bacterial, fungal, plant, or animalorigin, or fragments thereof), or radioactive isotopes.

In one embodiment, an immunoconjugate is an antibody-drug conjugate(ADC) in which an antibody is conjugated to one or more drugs, includingbut not limited to a maytansinoid (see U.S. Pat. Nos. 5,208,020,5,416,064 and EP 0 425 235 B1); an auristatin such as monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos.5,635,483, 5,780,588, and 7,498,298); a dolastatin; a calicheamicin orderivative thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116,5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman, L. M.et al., Cancer Res. 53 (1993) 3336-3342; and Lode, H. N. et al., CancerRes. 58 (1998) 2925-2928); an anthracycline such as daunomycin ordoxorubicin (see Kratz, F. et al., Curr. Med. Chem. 13 (2006) 477-523;Jeffrey, S. C. et al., Bioorg. Med. Chem. Lett. 16 (2006) 358-362;Torgov, M. Y. et al., Bioconjug. Chem. 16 (2005) 717-721; Nagy, A. etal., Proc. Natl. Acad. Sci. USA 97 (2000) 829-834; Dubowchik, G. M. etal., Bioorg. & Med. Chem. Letters 12 (2002) 1529-1532; King, H. D. etal., J. Med. Chem. 45 (20029 4336-4343; and U.S. Pat. No. 6,630,579);methotrexate; vindesine; a taxane such as docetaxel, paclitaxel,larotaxel, tesetaxel, and ortataxel; a trichothecene; and CC1065.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to an enzymatically active toxin or fragmentthereof, including but not limited to diphtheria A chain, nonbindingactive fragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to a Pseudomonas exotoxin A or variantsthereof. Pseudomonas exotoxin A or variants thereof are described e.g.in WO2011/32022, WO2009/32954, WO2007/031741, WO2007/016150,WO2005/052006 and Liu W, et al, PNAS 109 (2012) 11782-11787.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to a radioactive atom to form aradioconjugate. A variety of radioactive isotopes are available for theproduction of radioconjugates. Examples include At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰,Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu.When the radioconjugate is used for detection, it may comprise aradioactive atom for scintigraphic studies, for example TC^(99m) orI¹²³, or a spin label for nuclear magnetic resonance (NMR) imaging (alsoknown as magnetic resonance imaging, MRI), such as iodine-123 again,iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17,gadolinium, manganese or iron.

Conjugates of an antibody and cytotoxic agent may be made a) eitherusing recombination expression techniques (e.g. for the expression ofamino acid sequence based toxines fused to a Fab or Fv antibody fragmente.g. in E. coli) or b) using polypeptide coupling techniques (likesortase enzyme based coupling of amino acid sequence based toxines to aFab or Fv antibody fragment) or c) using a variety of bifunctionalprotein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctionalderivatives of imidoesters (such as dimethyl adipimidate HCl), activeesters (such as disuccinimidyl suberate), aldehydes (such asglutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta, E. S. et al., Science 238 (1987)1098-1104. Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriamine pentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO 94/11026. Thelinker may be a “cleavable linker” facilitating release of a cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari, R. V. et al., Cancer Res. 52 (1992)127-131; U.S. Pat. No. 5,208,020) may be used.

The immunuoconjugates or ADCs herein expressly contemplate, but are notlimited to such conjugates prepared with cross-linker reagentsincluding, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS,MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).

E. Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the anti-HER3/HER4 antibodies (or antigenbinding proteins) provided herein is useful for detecting the presenceof HER3 and/or HER4, respectively in a biological sample. The term“detecting” as used herein encompasses quantitative or qualitativedetection. In certain embodiments, a biological sample comprises a cellor tissue, such as tumor tissues.

In one embodiment, an anti-HER3/HER4 antibody for use in a method ofdiagnosis or detection is provided. In a further aspect, a method ofdetecting the presence of HER3 or HER4, respectively, in a biologicalsample is provided. In certain embodiments, the method comprisescontacting the biological sample with an anti-HER3/HER4 antibody asdescribed herein under conditions permissive for binding of theanti-HER3/HER4 antibody to HER3 or HER4, respectively, and detectingwhether a complex is formed between the anti-HER3/HER4 antibody and HER3or HER4, respectively. Such method may be an in vitro or in vivo method.In one embodiment, an anti-HER3/HER4 antibody is used to select subjectseligible for therapy with an the anti-HER3/HER4 antibodies antibody,e.g. where HER3 and HER4, respectively are both biomarkers for selectionof patients.

Exemplary disorders that may be diagnosed using an antibody of theinvention include cancer.

In certain embodiments, labeled anti-HER3/HER4 antibodies are provided.Labels include, but are not limited to, labels or moieties that aredetected directly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction. Exemplary labels include,but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels, stable free radicals, and the like.

F. Pharmaceutical Formulations

Pharmaceutical formulations of an anti-HER3/HER4 antibody (or antigenbinding protein) as described herein are prepared by mixing suchantibody having the desired degree of purity with one or more optionalpharmaceutically acceptable carriers (Remington's PharmaceuticalSciences, 16th edition, Osol, A. (ed.) (1980)), in the form oflyophilized formulations or aqueous solutions. Pharmaceuticallyacceptable carriers are generally nontoxic to recipients at the dosagesand concentrations employed, and include, but are not limited to:buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyl dimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspoly(vinylpyrrolidone); amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Exemplarypharmaceutically acceptable carriers herein further include interstitialdrug dispersion agents such as soluble neutral-active hyaluronidaseglycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidaseglycoproteins, such as rhuPH20 (HYLENEX®, Baxter International, Inc.).Certain exemplary sHASEGPs and methods of use, including rhuPH20, aredescribed in US Patent Publication Nos. 2005/0260186 and 2006/0104968.In one aspect, a sHASEGP is combined with one or more additionalglycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO 2006/044908, the latter formulationsincluding a histidine-acetate buffer.

The formulation herein may also contain more than one active ingredientsas necessary for the particular indication being treated.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methyl methacrylate) microcapsules, respectively, in colloidaldrug delivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th edition, Osol, A. (ed.) (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

G. Therapeutic Methods and Compositions

Any of the anti-HER3/HER4 antibodies (or antigen binding proteins) orimmunoconjugates of the anti-HER3/HER4 antibodies (or antigen bindingproteins) conjugated to a cytotoxic agent, provided herein may be usedin therapeutic methods.

In one aspect, an anti-HER3/HER4 antibody or immunoconjugate of theanti-HER3/HER4 antibody conjugated to a cytotoxic agent for use as amedicament is provided. In further aspects, an anti-HER3/HER4 antibodyor immunoconjugate of the anti-HER3/HER4 antibody conjugated to acytotoxic agent for use in treating cancer is provided. In certainembodiments, an anti-HER3/HER4 antibody or immunoconjugates of theanti-HER3/HER4 antibody conjugated to a cytotoxic agent for use in amethod of treatment is provided. In certain embodiments, the inventionprovides an anti-HER3/HER4 antibody or immunoconjugate of theanti-HER3/HER4 antibody conjugated to a cytotoxic agent for use in amethod of treating an individual having cancer comprising administeringto the individual an effective amount of the anti-HER3/HER4 antibody orthe immunoconjugate of the anti-HER3/HER4 antibody conjugated to acytotoxic agent. In further embodiments, the invention provides ananti-HER3/HER4 antibody or immunoconjugate of the anti-HER3/HER4antibody conjugated to a cytotoxic agent for use in inducing apoptosisin a cancer cell/or inhibiting cancer cell proliferation. In certainembodiments, the invention provides an anti-HER3/HER4 antibody orimmunoconjugate of the anti-HER3/HER4 antibody conjugated to a cytotoxicagent for use in a method of inducing apoptosis in a cancer cell/orinhibiting cancer cell proliferation in an individual comprisingadministering to the individual an effective of the the anti-HER3/HER4antibody or immunoconjugate of the anti-HER3/HER4 antibodies conjugatedto a cytotoxic agent to induce apoptosis in a cancer cell/or to inhibitcancer cell proliferation. An “individual” according to any of the aboveembodiments is preferably a human.

In a further aspect, the invention provides for the use of ananti-HER3/HER4 antibody or an immunoconjugate of the anti-HER3/HER4antibody conjugated to a cytotoxic agent in the manufacture orpreparation of a medicament. In one embodiment, the medicament is fortreatment of cancer. In a further embodiment, the medicament is for usein a method of treating cancer comprising administering to an individualhaving cancer an effective amount of the medicament. In a furtherembodiment, the medicament is for for inducing apoptosis in a cancercell/or inhibiting cancer cell proliferation. In a further embodiment,the medicament is for use in a method of inducing apoptosis in a cancercell/or inhibiting cancer cell proliferation in an individual sufferingfrom cancer comprising administering to the individual an amounteffective of the medicament to induce apoptosis in a cancer cell/or toinhibit cancer cell proliferation. An “individual” according to any ofthe above embodiments may be a human.

In a further aspect, the invention provides a method for treatingcancer. In one embodiment, the method comprises administering to anindividual having cancer an effective amount of an anti-HER3/HER4antibody. An “individual” according to any of the above embodiments maybe a human.

In a further aspect, the invention provides a method for inducingapoptosis in a cancer cell/or inhibiting cancer cell proliferation in anindividual suffering from cancer. In one embodiment, the methodcomprises administering to the individual an effective amount of ananti-HER3/HER4 antibody or an immunoconjugate of the anti-HER3/HER4antibody conjugated to a cytotoxic compound to induce apoptosis in acancer cell/or to inhibit cancer cell proliferation in the individualsuffering from cancer. In one embodiment, an “individual” is a human.

In a further aspect, the invention provides pharmaceutical formulationscomprising any of the anti-HER3/HER4 antibodies provided herein, e.g.,for use in any of the above therapeutic methods. In one embodiment, apharmaceutical formulation comprises any of the anti-HER3/HER4antibodies provided herein and a pharmaceutically acceptable carrier.

An antibody of the invention (and any additional therapeutic agent) canbe administered by any suitable means, including parenteral,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

Antibodies of the invention would be formulated, dosed, and administeredin a fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody of the invention (when used alone or in combination with one ormore other additional therapeutic agents) will depend on the type ofdisease to be treated, the type of antibody, the severity and course ofthe disease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments. Depending on the type and severityof the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.5 mg/kg-10 mg/kg) ofantibody can be an initial candidate dosage for administration to thepatient, whether, for example, by one or more separate administrations,or by continuous infusion. One typical daily dosage might range fromabout 1 μg/kg to 100 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment would generally be sustaineduntil a desired suppression of disease symptoms occurs. One exemplarydosage of the antibody would be in the range from about 0.05 mg/kg toabout 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg,4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administeredto the patient. Such doses may be administered intermittently, e.g.every week or every three weeks (e.g. such that the patient receivesfrom about two to about twenty, or e.g. about six doses of theantibody). An initial higher loading dose, followed by one or more lowerdoses may be administered. An exemplary dosing regimen comprisesadministering an initial loading dose of about 4 mg/kg, followed by aweekly maintenance dose of about 2 mg/kg of the antibody. However, otherdosage regimens may be useful. The progress of this therapy is easilymonitored by conventional techniques and assays.

It is understood that any of the above formulations or therapeuticmethods may be carried out using an immunoconjugate of the invention inplace of or in addition to an anti-HER3/HER4 antibody.

III. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an antibody of the invention. The label or package insertindicates that the composition is used for treating the condition ofchoice. Moreover, the article of manufacture may comprise (a) a firstcontainer with a composition contained therein, wherein the compositioncomprises an antibody of the invention; and (b) a second container witha composition contained therein, wherein the composition comprises afurther cytotoxic or otherwise therapeutic agent. The article ofmanufacture in this embodiment of the invention may further comprise apackage insert indicating that the compositions can be used to treat aparticular condition. Alternatively, or additionally, the article ofmanufacture may further comprise a second (or third) containercomprising a pharmaceutically-acceptable buffer, such as bacteriostaticwater for injection (BWFI), phosphate-buffered saline, Ringer's solutionand dextrose solution. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

It is understood that any of the above articles of manufacture mayinclude an immunoconjugate of the invention in place of or in additionto an anti-HER3/HER4 antibody.

Description of the Amino Acid Sequences

-   SEQ ID NO: 1 β-Hairpin of human HER3-   SEQ ID NO: 2 β-Hairpin of human HER4-   SEQ ID NO: 3 human HER3-   SEQ ID NO: 4 human HER3 Extracellular Domain (ECD)-   SEQ ID NO: 5 human HER4-   SEQ ID NO: 6 human HER4 Extracellular Domain (ECD)-   SEQ ID NO: 7 human HER1-   SEQ ID NO: 8 human HER1 Extracellular Domain (ECD)-   SEQ ID NO: 9 human HER2-   SEQ ID NO: 10 human HER2 Extracellular Domain (ECD)-   SEQ ID NO: 11 Human Heregulin fragment (HRG)-   SEQ ID NO: 12 Human Heregulin β-1 fragment (as provided from    Preprotech)-   SEQ ID NO: 13 TtSlyD-FKBP-Her3-   SEQ ID NO: 14 TtSlyD-Wildtype-   SEQ ID NO: 15 TtSlyDcas-   SEQ ID NO: 16 TgSlyDΔIF-   SEQ ID NO: 17 TtSlyDcas-Her3-   SEQ ID NO: 18 TtSlyDcys-Her3-   SEQ ID NO: 19 TgSlyDser-Her3-   SEQ ID NO: 20 TgSlyDcys-Her3-   SEQ ID NO: 21 TtSlyDcas-Her4-   SEQ ID NO: 22 TtSlyDcys-Her4-   SEQ ID NO: 23 TgSlyDser-Her4-   SEQ ID NO: 24 TgSlyDcys-Her4-   SEQ ID NO: 25 heavy chain HVR-H1, M-05-74-   SEQ ID NO: 26 heavy chain HVR-H2, M-05-74-   SEQ ID NO: 27 heavy chain HVR-H3, M-05-74-   SEQ ID NO: 28 light chain HVR-L1, M-05-74-   SEQ ID NO: 29 light chain HVR-L2, M-05-74-   SEQ ID NO: 30 light chain HVR-L3, M-05-74-   SEQ ID NO: 31 heavy chain variable domain VH, M-05-74-   SEQ ID NO: 32 light chain variable domain VL, M-05-74-   SEQ ID NO: 33 humanized variant 1 of heavy chain variable domain VH,    M-05-74_VH1-   SEQ ID NO: 34 humanized variant 2 of heavy chain variable domain VH,    M-05-74_VH2-   SEQ ID NO: 35 humanized variant 3 of heavy chain variable domain VH,    M-05-74_VH3-   SEQ ID NO: 36 humanized variant 1 of light chain variable domain VL,    M-05-74_VL1-   SEQ ID NO: 37 humanized variant 2 of light chain variable domain VL,    M-05-74_VL2-   SEQ ID NO:38 humanized variant 1 of HVR-H1, M-05-74_HVR-H1_V1-   SEQ ID NO:39 humanized variant 1 of HVR-H2, M-05-74_HVR-H2_V1-   SEQ ID NO:40 humanized variant 1 of HVR-L1, M-05-74_HVR-L1_V1-   SEQ ID NO: 41 humanized variant 1 of HVR-L2, M-05-74_HVR-L2_V1-   SEQ ID NO:42 humanized variant 2 of HVR-L2, M-05-74_HVR-L2_V2-   SEQ ID NO:43 binding epitope within β-hairpin of human HER3-   SEQ ID NO:44 binding epitope within β-hairpin of human HER4-   SEQ ID NO:45 Pseudomonas exotoxin variant PE24LR8M_3G (including a    GGG linker)-   SEQ ID NO:46 Light chain of M-05-74 (M-05-74_LC)-   SEQ ID NO:47 Heavy chain of M-05-74 HC with sortase tag (M-05-74_HC)-   SEQ ID NO:48 Heavy chain of M-05-74 HC conjugated to Pseudomonas    exotoxin variant PE24LR8M (Fab-074-PE heavy chain 1)-   SEQ ID NO:49 Heavy chain of M-05-74 HC conjugated to Pseudomonas    exotoxin variant PE24LR8M (Fab-074-PE heavy chain 2) as direct    PE24LR8M fusion-   SEQ ID NO: 50 soluble S. aureus sortase A-   SEQ ID NO: 51 heavy chain variable domain VH, <Her3> M-08-11-   SEQ ID NO: 52 light chain variable domain VL, <Her3> M-08-11-   SEQ ID NO: 53 human kappa light chain constant region-   SEQ ID NO: 54 human lambda light chain constant region-   SEQ ID NO: 55 human heavy chain constant region derived from IgG1-   SEQ ID NO: 56 human heavy chain constant region derived from IgG1    mutated on L234A and L235A-   SEQ ID NO: 57 human heavy chain constant region derived from IgG1    mutated on L234A, L235A and P329G-   SEQ ID NO: 58 human heavy chain constant region derived from IgG4

The following examples and figures are provided to aid the understandingof the present invention, the true scope of which is set forth in theappended claims. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.

In the following several embodiments of the invention are listed:

-   1. A method for selecting an antigen binding protein that binds to    human HER3 and binds to human HER4, wherein the antigen binding    protein binds within an amino acid sequence of PQPLVYNKLTFQLEPNPHT    (SEQ ID NO:1) of human HER3 and binds within an amino acid sequence    of PQTFVYNPTTFQLEHNFNA (SEQ ID NO:2) of human HER4;    -   wherein        -   a) at least one polypeptide selected from the group            consisting of:

SEQ ID NO: 13 TtSlyD-FKBP-Her3, SEQ ID NO: 17 TtSlyDcas-Her3, SEQ ID NO:18 TtSlyDcys-Her3, SEQ ID NO: 19 TgSlyDser-Her3, and SEQ ID NO: 20TgSlyDcys-Her3,

-   -   -   which comprises the amino acid sequence of SEQ ID NO:1;        -   and        -   b) at least one polypeptide selected from the group            consisting of:

SEQ ID NO: 21 TtSlyDcas-Her4, SEQ ID NO: 22 TtSlyDcys-Her4, SEQ ID NO:23 TgSlyDser-Her4, and SEQ ID NO: 24 TgSlyDcys-Her4,

-   -   -   which comprises the amino acid sequence of SEQ ID NO:2;

    -   are used to select antigen binding proteins, which show binding        to both, the at least one polypeptide under a) and the at least        one polypeptide under b)

    -   and thereby selecting an antigen binding protein that binds        within an amino acid sequence of PQPLVYNKLTFQLEPNPHT (SEQ ID        NO:1) and within an amino acid sequence of PQTFVYNPTTFQLEHNFNA        (SEQ ID NO:2).

-   2. An antigen binding protein obtained by the selection method of    embodiment 1.

-   3. The method of embodiment 1, or the antigen binding protein of    embodiment 2, wherein the antigen binding protein is an antibody.

-   4. An isolated antigen binding protein that binds to human HER3 and    binds to human HER4, wherein the antigen binding protein binds    within an amino acid sequence of PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1)    of human HER3 and binds within an amino acid sequence of    PQTFVYNPTTFQLEHNFNA (SEQ ID NO:2) of human HER4.

-   5. The antigen binding protein of embodiment 3    -   a) wherein the antigen binding protein binds to a polypeptide of

SEQ ID NO: 18 TtSlyDcys-Her3,

-   -    and    -   b) wherein the antigen binding protein binds to a polypeptide of

SEQ ID NO: 22 TtSlyDcys-Her4.

-   6. The antigen binding protein of embodiments 4 or 5 wherein the    antigen binding protein is an antibody.-   7. An isolated antibody that binds to human HER3 and that binds to    human HER4, wherein the antibody has one or more of the following    properties:    -   a) the antibody binds to the amino acid sequence of SEQ ID NO:1;        and/or    -   b) the antibody binds to the amino acid sequence SEQ ID NO:1 in        activated HER3; and/or    -   c) the antibody binds within an amino acid sequence of        PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1) which is comprised in a        polypeptide selected from the group consisting of:

SEQ ID NO: 13 TtSlyD-FKBP-Her3, SEQ ID NO: 17 TtSlyDcas-Her3, SEQ ID NO:18 TtSlyDcys-Her3, SEQ ID NO: 19 TgSlyDser-Her3, and SEQ ID NO: 20TgSlyDcys-Her3;

-   -    and/or    -   d) the antibody binds to the β-hairpin region of HER3; and/or    -   e) the antibody inhibits the heterodimerisation of HER3/HER2        heterodimers; and/or    -   f) the antibody binds to HER3-ECD with a ratio of the        association constant (Ka) in presence of Heregulin (Ka        (+Heregulin)) and in absence of Heregulin (Ka (−Heregulin)) of        4.0 or higher (Ka (+Heregulin))/(Ka (−Heregulin)); and/or    -   g) the antibody binds to HER3-ECD with a ratio of the Molar        Ratio MR of binding in presence of Heregulin (MR (+Heregulin))        and in absence of Heregulin (MR (−Heregulin)) of 2.0 or higher        (MR (+Heregulin))/(MR (−Heregulin)); and/or    -   h) the antibody binds to the amino acid sequence of SEQ ID NO:2;        and/or    -   i) the antibody binds to the amino acid sequence SEQ ID NO:2 in        activated HER4; and/or    -   j) the antibody binds within an amino acid sequence of        PQTFVYNPTTFQLEHNFNA (SEQ ID NO:2) which is comprised in a        polypeptide selected from the group consisting of:

SEQ ID NO: 21 TtSlyDcas-Her4, SEQ ID NO: 22 TtSlyDcys-Her4, SEQ ID NO:23 TgSlyDser-Her4, and SEQ ID NO: 24 TgSlyDcys-Her4;

-   -    and/or    -   k) the antibody binds to the β-hairpin region of HER4; and/or    -   l) the antibody binds to HER4-ECD with a ratio of the        association constant (Ka) in presence of Heregulin (Ka        (+Heregulin)) and in absence of Heregulin (Ka (−Heregulin)) of        20.0 or higher (Ka (+Heregulin))/(Ka (−Heregulin)); and/or    -   m) the antibody binds to HER4-ECD with a ratio of the Molar        Ratio MR of binding in presence of Heregulin (MR (+Heregulin))        and in absence of Heregulin (MR (−Heregulin)) of 5.0 or higher        (MR (+Heregulin))/(MR (−Heregulin)); and/or    -   n) the antibody shows as monovalent Fab fragment the same or        higher biological activity as compared to its bivalent parent        full length antibody; and/or    -   o) the antibody inhibits the HER3 phosporylation in MCF-7 cells;        and/or    -   p) the antibody does not compete for binding to HER3 with        Heregulin/induces binding of Heregulin to HER3; and/or    -   q) the antibody inhibits the proliferation of MDA-MB-175 tumor        cells; and/or    -   r) the antibody shows tumor growth inhibitory activity in vivo;        and/or    -   s) the antibody binds with an affinity of a KD value ≤1×10⁻⁸M to        HER3-ECD; and/or    -   t) the antibody binds with an affinity of a KD value ≤1×10⁻⁸M to        HER4-ECD; and/or    -   u) the antibody binds to a polypeptide consisting of VYNKLTFQLEP        (SEQ ID NO:43) and to a polypeptide consisting of VYNPTTFQLE        (SEQ ID NO:44); and/or    -   v) the antibody binds to a polypeptide consisting of VYNKLTFQLEP        (SEQ ID NO:43); and/or    -   w) the antibody binds to a polypeptide consisting of VYNPTTFQLE        (SEQ ID NO:44); and/or    -   x) the antibody binds in a FACS assay to HER3 expressing T47D        cells; and wherein the antibody shows an at least 25% higher        percentage of internalization in the presence of Heregulin as        compared to the percentage of internalization in the presence of        Heregulin when measured after 1 h after antibody exposure.

-   8. An antibody that binds to human HER3 and that binds human HER4,    wherein the antibody binds to a polypeptide with a length of 15    amino acids comprising the amino acid sequence VYNKLTFQLEP (SEQ ID    NO:43) (of human HER3) and to a polypeptide with a length of 15    amino acids comprising the amino acid sequence VYNPTTFQLE (SEQ ID    NO:44) (of human HER4).

-   9. The antibody of any one of embodiments 6 to 8, which is a human,    humanized, or chimeric antibody.

-   10. The antibody of any one of embodiments 6 to 8, which is an    antibody fragment that binds human HER3 and that binds human HER4.

-   11. The antibody of any one of embodiments 6 to 8, wherein the    antibody comprises (a) HVR-H1 comprising the amino acid sequence of    SEQ ID NO:25; (b) HVR-H2 comprising the amino acid sequence of SEQ    ID NO:26, and (c) HVR-H3 comprising the amino acid sequence of SEQ    ID NO:27.

-   12. The antibody of any one of embodiments 6 to 8, or 11,    comprising (a) HVR-L1 comprising the amino acid sequence of SEQ ID    NO:28; (b) HVR-L2 comprising the amino acid sequence of SEQ ID    NO:29; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID    NO:30.

-   13. The antibody of any one of embodiments 6 to 8, wherein the    antibody comprises    -   i) (a) HVR-H1 comprising the amino acid sequence of SEQ ID        NO:25;    -   (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:26;    -   (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:27;    -   (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:28;    -   (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:29;        and    -   (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:30;    -   ii) or a humanized variant of the HVRs of the antibody under i)        (a), (b), (d) and/or (e).

-   14. The antibody of any one of embodiments 6 to 8 or 13, wherein the    antibody comprises    -   i) (a) HVR-H1 comprising the amino acid sequence of SEQ ID        NO:38;    -   (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:39;    -   (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:27;    -   (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:40;    -   (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:41;        and    -   (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:30;        or    -   ii) (a) HVR-H1 comprising the amino acid sequence of SEQ ID        NO:38;    -   (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:39;    -   (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:27;    -   (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:40;    -   (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:42;        and    -   (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:30;        or    -   iii) (a) HVR-H1 comprising the amino acid sequence of SEQ ID        NO:25;    -   (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:39;    -   (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:27;    -   (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:40;    -   (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:41;        and    -   (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:30;        or    -   iv) (a) HVR-H1 comprising the amino acid sequence of SEQ ID        NO:25;    -   (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:39;    -   (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:27;    -   (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:40;    -   (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:42;        and    -   (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:30.

-   15. The antibody of any one of embodiments 6 to 8, comprising (a) a    VH sequence having at least 95% sequence identity to the amino acid    sequence of SEQ ID NO:33; (b) a VL sequence having at least 95%    sequence identity to the amino acid sequence of SEQ ID NO:36; or (c)    a VH sequence as in (a) and a VL sequence as in (b).

-   16. The antibody of embodiment 15, comprising a VH sequence of SEQ    ID NO: 33.

-   17. The antibody of embodiment 135, comprising a VL sequence of SEQ    ID NO: 36.

-   18. An antibody comprising a VH sequence of SEQ ID NO:33 and a VL    sequence of SEQ ID NO:36.

-   19. The antibody of any one of embodiments 6 to8, comprising (a) a    VH sequence having at least 95% sequence identity to the amino acid    sequence of SEQ ID NO:33; (b) a VL sequence having at least 95%    sequence identity to the amino acid sequence of SEQ ID NO:37; or (c)    a VH sequence as in (a) and a VL sequence as in (b).

-   20. The antibody of embodiment 19, comprising a VH sequence of SEQ    ID NO: 33.

-   21. The antibody of embodiment 19, comprising a VL sequence of SEQ    ID NO: 37.

-   22. An antibody comprising a VH sequence of SEQ ID NO:33 and a VL    sequence of SEQ ID NO:37.

-   23. The antibody of any one of embodiments 6 to 8, comprising (a) a    VH sequence having at least 95% sequence identity to the amino acid    sequence of SEQ ID NO:34; (b) a VL sequence having at least 95%    sequence identity to the amino acid sequence of SEQ ID NO:36; or (c)    a VH sequence as in (a) and a VL sequence as in (b).

-   24. The antibody of embodiment 23, comprising a VH sequence of SEQ    ID NO: 34.

-   25. The antibody of embodiment 23, comprising a VL sequence of SEQ    ID NO: 36.

-   26. An antibody comprising a VH sequence of SEQ ID NO:34 and a VL    sequence of SEQ ID NO:36.

-   27. The antibody of any one of embodiments 6 to 8, comprising (a) a    VH sequence having at least 95% sequence identity to the amino acid    sequence of SEQ ID NO:34; (b) a VL sequence having at least 95%    sequence identity to the amino acid sequence of SEQ ID NO:37; or (c)    a VH sequence as in (a) and a VL sequence as in (b).

-   28. The antibody of embodiment 27, comprising a VH sequence of SEQ    ID NO: 34.

-   29. The antibody of embodiment 27, comprising a VL sequence of SEQ    ID NO: 37.

-   30. An antibody comprising a VH sequence of SEQ ID NO:34 and a VL    sequence of SEQ ID NO:37.

-   31. The antibody of any one of embodiments 6 to 8, comprising (a) a    VH sequence having at least 95% sequence identity to the amino acid    sequence of SEQ ID NO:35; (b) a VL sequence having at least 95%    sequence identity to the amino acid sequence of SEQ ID NO:36; or (c)    a VH sequence as in (a) and a VL sequence as in (b).

-   32. The antibody of embodiment 31, comprising a VH sequence of SEQ    ID NO: 35.

-   33. The antibody of embodiment 31, comprising a VL sequence of SEQ    ID NO: 36.

-   34. An antibody comprising a VH sequence of SEQ ID NO:35 and a VL    sequence of SEQ ID NO:36.

-   35. The antibody of any one of embodiments 6 to 8, comprising (a) a    VH sequence having at least 95% sequence identity to the amino acid    sequence of SEQ ID NO:35; (b) a VL sequence having at least 95%    sequence identity to the amino acid sequence of SEQ ID NO:37; or (c)    a VH sequence as in (a) and a VL sequence as in (b).

-   36. The antibody of embodiment 35, comprising a VH sequence of SEQ    ID NO: 35.

-   37. The antibody of embodiment 35, comprising a VL sequence of SEQ    ID NO: 37.

-   38. An antibody comprising a VH sequence of SEQ ID NO:35 and a VL    sequence of SEQ ID NO:37.

-   39. The antibody of any one of embodiments6 to 38, which is a full    length IgG1 antibody or IgG4 antibody.

-   40. The antibody of any one of embodiments 6 to 38, which is a Fab    fragment.

-   41. Isolated nucleic acid encoding the antibody of any one of    embodiments 6 to 38.

-   42. A host cell comprising the nucleic acid of embodiment41.

-   43. A method of producing an antibody comprising culturing the host    cell of embodiment 42 so that the antibody is produced, and    recovering said antibody from said cell culture or the cell culture    supernatant.

-   44. An immunoconjugate comprising the antibody of any one of    embodiments 6 to 38 and a cytotoxic agent.

-   45. A pharmaceutical formulation comprising the antibody of any one    of embodiments 6 to 38, or the immunoconjugate of embodiment 44, and    a pharmaceutically acceptable carrier.

-   46. The antibody of any one of embodiments 6 to 38, or the    immunoconjugate of embodiment 44, for use as a medicament.

-   47. The antibody of any one of embodiments 6 to 38, or the    immunoconjugate of embodiment 44, for use in treating cancer.

-   48. The antibody of any one of embodiments 6 to 38 for use in    inhibition of HER3/HER2 dimerization.

-   49. Use of the antibody of any one of embodiments 6 to 38, or an    immunoconjugate of embodiment 44, in the manufacture of a    medicament.

-   50. The use of embodiment 49, wherein the medicament is for    treatment of cancer.

-   51. The use of embodiment 49, wherein the medicament is for the    inhibition of HER3/HER2 dimerization.

-   52. A method of treating an individual having cancer comprising    administering to the individual an effective amount of the antibody    of any one of the preceding embodiments, or an immunoconjugate    comprising the antibody of any one of the preceding embodiments and    a cytotoxic agent.

-   53. A method of inducing apoptosis in a cancer cell in an individual    suffering from cancer comprising administering to the individual an    effective amount of an immunoconjugate comprising the antibody of    any one of the preceding embodiments and a cytotoxic agent, thereby    inducing apoptosis in a cancer cell in the individual.

-   54. A polypeptide selected from the group consisting of:

i) SEQ ID NO: 13 TtSlyD-FKBP-Her3, ii) SEQ ID NO: 17 TtSlyDcas-Her3,iii) SEQ ID NO: 18 TtSlyDcys-Her3, iv) SEQ ID NO: 19 TgSlyDser-Her3, andv) SEQ ID NO: 20 TgSlyDcys-Her3,

-   -   which polypeptide comprises the amino acid sequence of SEQ ID        NO:1

-   55. A polypeptide selected from the group consisting of:

i) SEQ ID NO: 21 TtSlyDcas-Her4, ii) SEQ ID NO: 22 TtSlyDcys-Her4, iii)SEQ ID NO: 23 TgSlyDser-Her4, and iv) SEQ ID NO: 24 TgSlyDcys-Her4,

-   -   which polypeptide comprises the amino acid sequence of SEQ ID        NO:2

EXAMPLES

Materials & General Methods

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook,J. et al., Molecular Cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. The molecularbiological reagents were used according to the manufacturer'sinstructions.

Gene Synthesis

Desired gene segments were prepared from oligonucleotides made bychemical synthesis. The 400-1600 bp long gene segments, which wereflanked by singular restriction endonuclease cleavage sites, wereassembled by annealing and ligating oligonucleotides including PCRamplification and subsequently cloned via the indicated restrictionsites e.g. EcoRI/BlpI or BsmI/XhoI into the expression vectors describedbelow. The DNA sequences of the subcloned gene fragments were confirmedby DNA sequencing. Gene synthesis fragments were ordered according togiven specifications at Geneart (Regensburg, Germany).

DNA Sequence Determination

DNA sequences were determined by double strand sequencing performed atSequiserve GmbH (Vaterstetten, Germany).

DNA and Protein Sequence Analysis and Sequence Data Management

Infomax's Vector NT1 Advance suite version 11.5.0 was used for sequencecreation, mapping, analysis, annotation and illustration.

Example 1

Preparation of Antigen and Screening Proteins—Generation of Functionalβ-hairpin HER3 and β-hairpin HER4 Constructs for Selecting AntibodiesBinding to the β-hairpin of HER3 and the β-hairpin of HER4

To generate functional β-Hairpin HER3 and HER4 constructs, the aminoacid sequences of the β-Hairpins of HER3 (SEQ ID NO: 1) and HER4 (SEQ IDNO: 2), were grafted into a SlyD polypeptide framework comprising a FKBPdomain. In such constructs the grafted β-Hairpins are freely accessiblein contrast to the hidden structure in the native unactivatedconformation of HER3 or HER4 (in the absence of ligand as e.g. HRG) (seeFIGS. 1C and 1D where the β-Hairpin of HER3 is hidden).

All fused SlyD polypeptides can be purified and refolded by using almostidentical protocols. E. coli BL21 (DE3) cells transformed with theparticular expression plasmid were grown at 37° C. in LB mediumcontaining the respective antibiotic for selective growth (Kanamycin 30μg/ml, or Ampicillin (100 μg/ml)) to an OD600 of 1.5, and cytosolicoverexpression was induced by adding 1 mM isopropyl-β-D-thiogalactoside(IPTG). Three hours after induction, cells were harvested bycentrifugation (20 min at 5,000 g), frozen and stored at −20° C. Forcell lysis, the frozen pellet was resuspended in chilled 50 mM sodiumphosphate buffer (pH 8.0) supplemented with 7 M GdmCl and 5 mMimidazole. Thereafter the suspension was stirred for 2-10 hours on iceto complete cell lysis. After centrifugation (25,000 g, 1 h) andfiltration (cellulose nitrate membrane, 8.0 μm, 1.2 μm, 0.2 μm), thelysate was applied onto a Ni-NTA column equilibrated with the lysisbuffer. In the subsequent washing step the imidazole concentration wasraised to 10 mM (in 50 mM sodium phosphate buffer (pH 8.0) comprising 7M GdmCl) and 5 mM TCEP was added in order to keep the thiol moieties ina reduced form and to prevent premature disulfide bridging. At least 15to 20 volumes of the reducing washing buffer were applied. Thereafter,the GdmCl solution was replaced by 50 mM sodium phosphate buffer (pH8.0) comprising 100 mM NaCl, 10 mM imidazole, and 5 mM TCEP to induceconformational refolding of the matrix-bound SlyD fusion polypeptide. Inorder to avoid reactivation of co-purifying proteases, a proteaseinhibitor cocktail (Complete® EDTA-free, Roche) was added to therefolding buffer. A total of 15 to 20 column volumes of refolding bufferwere applied in an overnight procedure. Thereafter, both TCEP and theComplete® EDTA-free inhibitor cocktail were removed by washing with 10column volumes 50 mM sodium phosphate buffer (pH 8.0) comprising 100 mMNaCl and 10 mM imidazole. In the last washing step, the imidazoleconcentration was raised to 30 mM (10 column volumes) in order to removetenacious contaminants. The refolded polypeptide was then eluted byapplying 250 mM imidazole in the same buffer. Protein-containingfractions were assessed for purity by Tricine-SDS-PAGE (Schaegger, H.and von Jagow, G., Anal. Biochem. 166 (1987) 368-379). Subsequently, theprotein was subjected to size-exclusion-chromatography (Superdex™HiLoad, Amersham Pharmacia) using potassium phosphate as the buffersystem (50 mM potassium phosphate buffer (pH 7.0), 100 mM KCl, 0.5 mMEDTA). Finally, the protein-containing fractions were pooled andconcentrated in an Amicon cell (YM10) to a concentration of ˜5 mg/ml.Exemplarily SDS-PAGE analysis of Ni-NTA purification of TtSlyD-FKBP-Her3is shown in FIG. 3 and SEC elution profile of a Ni-NTA purified fractionof Thermus thermophilus SlyD-FKBP-Her-3 is shown in FIG. 4. The Thermusthermophilus SlyD (TtSlyD)-Her-3 fusion polypeptide could be purifiedsuccessfully as a soluble and stable polypeptide in its monomeric form.The final yield was quantified at 16.4 mg purified protein from fraction12 and 13.

Table 2: Summary of the amino acid sequences of the developed SlyD-basedepitope scaffolds (which carry the HER3 dimerization domain fragment(β-Hairpin of HER3 (SEQ ID NO: 1)) as insert or the HER4 dimerizationdomain fragment (β-Hairpin of HER4 (SEQ ID NO: 2)) as insert).

TtSlyD-FKBP-Her3, TtSlyDcas-Her3, TtSlyDcys-Her3, Thermococcusgammatolerans TgSlyDser-Her3 and TgSlyDcys-Her3 carry the Her3dimerization domain fragment (β-Hairpin of HER3 (SEQ ID NO: 1)) asinsert and were used as immunogens and as positive controls in ELISAscreening.

TtSlyD-Wildtype, TtSlyDcas, TgSlyDΔIF were used as negative controls inthe ELISA screening (without the Her3 dimerization domain fragment(β-Hairpin of HER3 (SEQ ID NO: 1)) or the Her4 dimerization domainfragment (β-Hairpin of HER4 (SEQ ID NO: 2)) as insert).

TtSlyDcas-Her4, TtSlyDcys-Her4, TgSlyDser-Her4 and TgSlyDcys-Her4 (whichcarry the Her4 dimerization domain fragment (β-Hairpin of HER4 (SEQ IDNO: 2)) as insert) were used in the ELISA screening to check thedeveloped clones for HER4 crossreactivity.

As the epitope scaffolds are expressed in E. coli the N-terminalmethionine residue can be present or not. (Nt=N-terminal; Ct=C-terminal)

TABLE 2 TtSlyD- Nt- FKBP- MRSKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGLEHer3 EALEGREEGEAFQAHVPAEKAYGAGSPQPLVYNKLTFQLEPNPHTKGSSGKDLDFQVEVVKVREATPEELLHGHAHG GGSRKHHHHHHHH-Ct TtSlyD- Nt- WildtypeMRGSKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGLEEALEGREEGEAFQAHVPAEKAYGPHDPEGVQVVPLSAFPEDAEVVPGAQFYAQDMEGNPMPLTVVAVEGEEVTVDFNHPLAGKDLDFQVEVVKVREATPEELLHGHAHGGGSRKHHHHHHHH-Ct TtSlyDcas Nt-MRSKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGLEEALEGREEGEAFQAHVPAEKAYGAGSGSSGKDLDFQVEVVKVREATPEELLHGHAHGGGSRKHHHHHHHH-Ct TgSlyDΔIF Nt-MKVERGDFVLFNYVGRYENGEVFDTSYESVAREQGIFVEEREYSPIGVTVGAGEIIPGIEEALLGMELGEKKEVVVPPEKGYGATGHPGIIPPHATAIFEIEVVEIKKAGEALEHHHHHHLEHHHHHH-Ct TtSlyDcas- Nt- Her3MRSKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGLEEALEGREEGEAFQAHVPAEKAYGAGSPQPLVYNKLTFQLEPNPHTKGSSGKDLDFQVEVVKVREATPEELLHGHAHGGGSRKHHH HHHHH-Ct TtSlyDcys- Nt- Her3MRGSKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGLEEALEGREEGEAFQAHVPAEKAYGPCGPQPLVYNKLTFQLEPNPHTGCGKDLDFQVEVVKVREATPEELLHGHAHGGGSHHHHHH HH-Ct TgSlyDser- Nt- Her3MKVERGDFVLFNYVGRYENGEVFDTSYESVAREQGIFVEEREYSPIGVTVGAGEIIPGIEEALLGMELGEKKEVVVPPEKGYGMPSGPQPLVYNKLTFQLEPNPHTGSAGKTAIFEIEVVEIKKAGEAGGG SRKHHHHHHHH-Ct TgSlyDcys-Nt- Her3 MRGSKVERGDFVLFNYVGRYENGEVFDTSYESVAREQGIFVEEREYSPIGVTVGAGEIIPGIEEALLGMELGEKKEVVVPPEKGYGMPCGPQPLVYNKLTFQLEPNPHTGCAGKTAIFEIEVVEIKKAGEA GGGSHHHHHHHH-Ct TtSlyDcas-Nt- Her4 MRSKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGLEEALEGREEGEAFQAHVPAEKAYGAGSPQTFVYNPTTFQLEHNFNAKGSSGKDLDFQVEVVKVREATPEELLHGHAHGGGSRKHHH HHHHH-Ct TtSlyDcys- Nt- Her4MRGSKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGLEEALEGREEGEAFQAHVPAEKAYGPCGPQTFVYNPTTFQLEHNFNAGCGKDLDFQVEVVKVREATPEELLHGHAHGGGSHHHHHH HH-Ct TgSlyDser- Nt- Her4MKVERGDFVLFNYVGRYENGEVFDTSYESVAREQGIFVEEREYSPIGVTVGAGEIIPGIEEALLGMELGEKKEVVVPPEKGYGMPSGPQTFVYNPTTFQLEHNFNAGSAGKTAIFEIEVV EIKKAGEAGGGSRKHHHHHHHH-CtTgSlyDcys- Nt-MRGSKVERGDFVLFNYVGRYENGEVFDTSYESVAREQGIFVEE Her4REYSPIGVTVGAGEIIPGIEEALLGMELGEKKEVVVPPEKGYGMPCGPQTFVYNPTTFQLEHNFNAGCAGKTAIFEIEVVEIKKAGEA GGGSHHHHHHHH-Ct

Example 2

a) Immunisation and Selection of HER3 Antibodies

For the generation of antibodies against the β-hairpin of HER3 and HER4,Balb/C, NMRI or SJL mice were immunized with different antigens. Asantigens the following proteins were used: full length Her3 ECD, or theepitope scaffold proteins TtSlyD-FKBP12-Her3, TtSlyDcys-Her3,TtSlyDcas-Her3, TgSlyDcys-Her3 and TgSlyDser-Her3. TheTtSlyD-FKBP12-Her3 variant represents the first generation epitopescaffold, used for generation of Her3 dimerization domain specificantibodies. Although the general principal of using SlyD variants asepitope scaffolds could already be demonstrated using the firstgeneration SlyD-FKBP12 scaffold, improved variants of the scaffold withhigher stability were developed. These SlyD variants are derived fromThermos thermophilus and Thermococcus gammatolerans.

All mice were subjected to 3 immunizations at the time points 0, 6 and10 weeks after start of the immunization campaign. At each time pointeach mouse was immunized with 100 μg endotoxin free immunogen dissolvedin 100 μl PBS. For the first immunization the immunogen was mixed with100 μl CFA. For the second and third immunization the immunogen wasmixed with IFA. The first and the third immunization were applied viathe intraperitoneal route, the second immunization was appliedsubcutaneously. 2 and 3 days prior to the preparation of spleenocyte forantibody development using hybridoma technology, the mice were subjectedto intravenous booster immunizations with 12.5 μg immunogen in 100 μlPBS and without adjuvant.

Titer Analysis

For the determination of serum titers against the respective immunogenand against the screening proteins a small amount of serum of each mousewas collected in week 11 after start of the immunization campaign. Forthe ELISA the immunogen or the screening scaffold proteins wereimmobilized on the plate surface. Her3 ECD was immobilized at aconcentration of 1 μg/ml and the scaffold proteins TtSlyD-FKBP12-Her3,TtSlyD-FKBP12, TtSlyDcys-Her3, TtSlyDcas-Her3, TtSlyDcas,TgSlyDcys-Her3, TgSlyDser-Her3 and TgSlyDΔIF were used at aconcentration of 0.5 μg/ml. The scaffold proteins TtSlyDcas andTgSlyDΔIF were used as negative controls. The sera from each mouse werediluted in PBS with 1% BSA and the dilutions were added to the plates.The sera were tested at dilutions 1:300, 1:900, 1:2700, 1:8100, 1:24300,1:72900, 1:218700 and 1:656100. Bound antibody was detected with aHRP-labeled F(ab′)₂ goat anti-mouse Fcγ (Dianova) and ABTS (Roche) as asubstrate.

Even on the level of serum titration it was already obvious thatimmunized mice developed antibodies against the Her3 β-hairpin domain.In mice immunized with Her3 ECD this can be shown by titration againstone of the scaffold proteins containing the dimerization β-hairpin loop.The strongly reduced signal can be explained by the fact, that themajority of antibodies raised by immunization with Her3 ECD aretargeting other parts within the ECD and only a small fraction isbinding to the dimerization β-hairpin domain. In mice immunized withHer3 dimerization loop containing scaffolds the fraction of antibodiestargeting the loop can be shown by titration against Her3 ECD (positivecontrol) and titration against an control scaffold without Her3insertion (negative control).

b) Antibody Development and ELISA Screening/Selection

The use of the here described epitope scaffold technology offers inprincipal two strategies for the development of antibodies targeting theHer3 dimerization domain (β-Hairpins of HER3 (SEQ ID NO: 1)). Onestrategy is to immunize with the full length Her3 ECD and to use thescaffolds to screen for the dimerization domain specific antibodies. Theother strategy is the direct use of the scaffold for immunization and touse the Her3 ECD, a scaffold with another backbone or a scaffold withoutinsertion for counter screening. Antibodies were developed withhybridoma technology by fusing primary B-cells with P3X63Ag8.653 myelomacells. 2 days after the final booster immunization, immunized mice weresacrificed and spleen cell populations were prepared. The spleenocyteswere fused with P3X63Ag8.653 by using the PEG fusion technology. Thecellular batch culture from the fusion was incubated overnight at 37° C.under 5% CO₂. The following day the cellular batch containing fusedcells was centrifuged for 10 min at 400 g. Thereafter, the cells weresuspended in hybridoma selection media supplemented with 0.1×azaserine-hypoxanthine (Sigma) and were seeded at a concentration of2.5×10⁴ cells per well in 96 well plates. The plates were cultured forat least 1 week at 37° C. under 5% CO₂. 3 days prior to ELISA analysisthe selection media was changed.

Primary culture supernatants were tested in ELISA against Her3 ECD andvarious scaffold proteins. The testing against the scaffold proteins wasdone to demonstrate that the selected clones are binding to thedimerization domain β-hairpin of native Her3 ECD. The testing againstthe control scaffolds TtSlyDcas and TgSlyDΔIF was done to show that theselected clones are binding the inserted Her3 derived sequence and notthe scaffold backbone. To check for cross reactivity the resultingclones were tested against the full length ECDs of the other members ofthe Her family namely, Her1, Her2 and Her4. As shown all selected clonesare highly specific for Her3 and a highly specific cross reactivity toHER4 could be detected, while no cross reactivity to other members ofthe Her family were detected. For the ELISA the screening an antigendown format was used. Her3 ECD was immobilized at a concentration of 1μg/ml and the scaffold proteins TtSlyD-FKBP12-Her3, TtSlyD-FKBP12,TtSlyDcys-Her3, TtSlyDcas-Her3, TtSlyDcas, TgSlyDcys-Her3,TgSlyDser-Her3 and TgSlyDΔIF were immobilized at a concentration of 0.5μg/ml. Hybridoma Supernatant was added to the plates and incubated for 1h at room temperature. Bound antibody was detected with a HRP-labeledF(ab′)₂ goat anti-mouse Fcγ (Dianova) and ABTS (Roche) was used as aHRP-substrate.

TABLE 3 Evaluation of the selected clones by ELISA. The clones weretested against the scaffold proteins TtSlyDcas-Her3, TtSlyDcys-Her3,TgSlyDser-Her3 and TgSlyDcys-Her3 and the full length Her3 ECD to verifytheir Her3 dimerization domain insert (ß-Hairpin of HER3 (SEQ ID NO: 1))specificity. As negative controls the scaffold proteins TtSlyDcas andTgSlyDΔIF were used. Additionally, clones were tested against fulllength ECDs of Her1, Her2, Her3 and Her4 to verify potential crossreactivity. Clones show binding to full length Her3 ECD and are crossreactive against full length Her4 ECD. TtSlyD- TgSlyD- cys- cys- Her1Her2 Her3 Her4 Klon cas cas-Her3 Her3 ΔIF ser-Her3 Her3 ECD ECD ECD ECDM-05- 0.023 3.133 3.150 0.020 3.159 3.159 0.018 0.020 3.152 3.170 74M-15- 0.040 1.763 1.522 0.040 1.980 1.785 0.024 0.025 3.153 3.192 02M-15- 0.045 1.772 1.850 0.039 1.628 1.461 0.020 0.024 3.171 3.234 03M-15- 0.040 1.847 1.457 0.033 1.833 1.500 0.067 0.064 3.175 3.186 04M-15- 0.041 1.443 1.482 0.046 1.886 1.485 0.020 0.021 3.156 3.216 05M-15- 0.041 1.569 1.707 0.040 1.746 1.532 0.019 0.023 3.195 3.181 08M-15- 0.057 1.870 1.929 0.076 1.799 1.640 0.024 0.037 3.234 3.200 09M-15- 0.044 1.714 1.636 0.056 2.005 1.693 0.029 0.031 3.103 3.218 11M-16- 0.039 1.653 1.793 0.037 1.860 1.637 0.024 0.032 3.184 3.212 01c) Immunohistochemistry

All selected clones were tested for reactivity and specificity in IHC.Therefore HEK293 cells were transiently transfected with plasmids codingfor full length HER1, HER2, HER3 or HER4, respectively. 2 days aftertransfection the different cell lines now expressing HER1, HER2, HER3 orHER4were harvested, subsequently fixed in formalin and embedded inAgarose for generation of IHC controls. After an additional fixation informalin overnight the Agarose blocks were embedded in paraffin.Untransfected HEK293 cells were used as negative controls and treatedaccordingly to the transfected cells. After paraffin embedding 3 μm thinsections were prepared using a microtome. The sections were mounted onglass microscopy slides and dried for 2 h. All further steps of theimmunohistochemical staining procedure were carried out using a VentanaBenchmark XT. The slides were dewaxed and antigen retrieval wasperformed by applying heat for 1 hour. For antigen retrieval the Ventanabuffer CC1 was used. The antibodies were used at a concentration of 1μg/ml. For the detection of bound antibody the Ventana UltraViewdetection kit was used. Results are shown in FIG. 5. All three clonesshowed binding to HER3 and cross reactivity against HER4. No crossreactivity against HER1 and HER2 was detectable.

d) DNA Sequencing of Selected Anti-Her3 Hybridoma

To obtain the DNA sequences of the selected hybridoma clones a 5′ RacePCR was conducted. For the RT-PCR total RNA was prepared from 5×10⁶cells by using a total RNA purification kit (Qiagen). The reversetranscription and the PCR were conducted using a 5′ prime RACE PCR kit(Roche). The resulting PCR fragments from heavy and light chain werepurified by gel electrophoresis and subsequent gel purification. The PCRfragments were cloned using the Topo Zero-Blunt cloning kit (Invitrogen)and transformed into competent cells. Several clones from each hybridomawere submitted for sequencing to obtain a consensus sequences for theselected clones. M-05-74 M-15-02 M-15-04 were submitted for sequencingwhich resulted in identical VH and VL sequences for all 3 clones.M-15-03, M-15-05, M-15-08, M-15-09, M-15-11, M-16-01 were sequencedanalogously and also resulted in identical VH and VL sequences for allclones.

e) Time Dependent Internalization Analyses of M-05-74 via FACS

Binding and internalization of HER3 by the selected clone M-05-74 toHER3 was analyzed in FACS using the HER3 expressing tumor cell lineT47D. 5×10⁵ cells were treated with 50 ng Recombinant Human Heregulinfragment (HRG) (SEQ ID NO: 11). The fragment including amino acid of SEQID NO: 11 was cloned in pCDNA.1 vector (Invitrogen). The HRG fragmentwas expressed in FreeStyle™ 293-F cells according to the protocoldescribed by Invitrogen. (FreeStyle™ 293 Expression system Catalog no.K9000-01). Purified HRG fragment was solved in 20 mM Histidin,140 mMNaCl; pH6.0 and stored by −80 C.

Untreated (−) cells were used as negative controls. Shortly afterHeregulin induced activation, 1 μg of M-05-74 was added to the cells.The cells were incubated for 0, 5, 15, 30, 45, 60, 75, 90, 105, 120, 180or 240 min at 37° C. After incubation the cells were immediately put onice. The cells were washed with 3 ml FACS buffer once and then stainedfor 30 minutes with 1 μg of a R-Phycoerythrin Goat Anti-Mouse IgG (H+L)secondary antibody. Flow cytometry was carried out using a FACSCanto™flow cytometer (BD Biosciences). Results are FACS analysis of M-05-74induced, time dependent HER3 receptor internalization in T47D cells.M-05-74 shows binding to the expressed HER3 ECD, with or withoutsupplemented recombinant human Heregulin fragment (HRG). M-05-74 leadsto Her3 receptor internalization over a 4 h time period. Results areshown in FIG. 6. The isotype control is indicated as a constanthorizontal black bar. M-05-74 shows binding to the expressed Her3 ECD,with or without Human Heregulin fragment (−) and (+HRG). M-05-74 leadsto Her3 receptor internalization over a 4 h time period. The isotypecontrol is indicated as a constant horizontal black bar. In the presenceof HRG the antibody induced internalization of HER3 was faster (e.g.after 1 h, at least 25% more HER3 were internalized in the presence ofHRG (+HRG) when compared to the value in the absence of HRG (−).

Example 3

a) Kinetic Screening/Binding Properties of HER3 Antibodies

The kinetic screening was performed according to Schraeml et al.(Schraml, M. and M. Biehl, Methods Mol Biol 901 (2012) 171-181) on aBIAcore 4000 instrument, mounted with a Biacore CMS sensor. In all assaythe test antibodies were captured. The system was under the control ofthe software version V1.1. The instrument buffer was HBS-EP (10 mM HEPES(pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.05% (w/v) P20). The system operatedat 25° C. 30 μg/ml Rabbit polyclonal antibody (RAM IgG, (Rabbit antiMouse IgG with Fc gamma specificity) GE Healthcare) in 10 mM sodiumacetate buffer (pH 4.5) was immobilized using EDC/NHS chemistryaccording to the manufacturer's instructions on the spots 1, 2, 4 and 5in the flow cells 1, 2, 3 and 4. The sensor was saturated using 1Methanolamine. In each flow cell, referenced signals were calculatedusing spots 1-2 and spots 5-4, spot 3 served as a blanc control. Theantigen (human recombinant Her-3 ECD (68 kDa), and recombinant Thermusthermophilus SlyD FKBP-Her3 (15 kDa) comprising the β-hairpin peptide ofHER3 (SEQ ID NO:1)) was diluted at 150 nM in instrument buffersupplemented with 1 mg/ml CMD (Carboxymethyldextran, Sigma). to suppressunspecific binding. Prior to their application the hybridoma culturesupernatants were diluted 1:5 in instrument buffer. The diluted mixtureswere injected at a flow rate of 30 μl/min for 2 min. The antibodycapture level (CL) in response units was monitored. Immediatelythereafter the respective antigen was injected at a flow rate of 30μl/min for 3 min association time. Thereafter, the antibody-antigencomplex dissociation signal was recorded for 5 min. The sensor wasregenerated by injecting a 10 mM glycine-HCl solution (pH 1.7) for 2 minat a flow rate of 30 μl/min. The recorded signal shortly before the endof the injection of the antigen was denoted as binding late (BL) inresponse units. The recorded signal shortly before the end of therecording of the dissociation is denoted as stability late (SL) inresponse units. The dissociation rate constants were determinedcalculated The antibody-antigen complex stability in minutes wascalculated with the following formula: ln(2)/60*kd. The Molar Ratio wascalculated with the formula: MW (antibody)/MW(antigen) *BL (antigen)/CL(antibody).

Binding Late (BL) represents the response units at the end of theanalyte injection. The amount of antibody captured as a ligand on thesensor surface is measured as Capture Level (CL) in response units.Together with the information of the molecular weights of the testedanalytes, the antibody and the analyte in solution, the Molar Ratio canbe calculated. In case the sensor was configurated with a suitableamount of antibody ligand capture level, each antibody should be able tofunctionally bind at least to one analyte in solution, which isrepresented by a Molar Ratio of MR=1.0. Then, the Molar Ratio is also anindicator for the valence mode of analyte binding. The maximum valencecan be MR=2 for an antibody binding two analytes, one with each Fabvalence. In case of steric limitations or a dysfunctional analytebinding, the Molar Ratio can indicate understoichiometric binding, likeit is the case when the Her-3 ECD is being bound in its “closed”conformation. The maximum assay deviation in the determination of theMolar Ratio is MR=0.2.

Screening/Selection of Anti-HER3/HER4 Antibody M-05-74:

In one experiment, the kinetic screening was driven with hybridomaprimary cultures from different fusions, which were obtained from animmunization of mice with human recombinant Her-3 ECD. The aim was toselect cultures with binding specificity for the Her-3heterodimerization domain β-hairpin peptide (SEQ ID NO:1). As antigensin solution human recombinant Her-3 ECD (68 kDa), and recombinantThermus thermophilus SlyD FKBP-Her3 (15 kDa) comprising the β-hairpinpeptide of HER3 (SEQ ID NO:1) were used. A positive hit was classifiedas a primary culture supernatant with binding activity versus bothantigens.

The Table 4 exemplarily shows primary culture supernatants, from whichM-05-74 fulfills these requirements, indicating epitope specificity forthe β-hairpin of HER3. Therefore this is a suitable method of screeningof anti-HER3 antibodies which bind to the Her-3 hairpin of SEQ ID NO:1.

TABLE 4 Exemplary results obtained from a kinetic screening experimentwith a set of hybridoma primary cultures from fusions, wherein antibodyM-05-74 was identified as binding to both HER3 ECD and the ß-hairpin ofHER3 (SEQ ID NO: 1) within the thermo SlyD-Her3 construct. stabilitybinding late late BL SL kd t/2 diss T CL MR Ligand Analyte [RU] [RU][1/s] [min] [° C.] [RU] [—] M-04- human- 17 16 4.13E−04  28 25 134 0.306 Her3- ECD M-04- thermo −4 −4 n.d. n.d. 25 134 −0.3 06 SlyD- Her3M-04- human- −1 1 n.d. n.d. 25 110 0.0 140 Her3- ECD M-04- thermo −6 −5n.d. n.d. 25 112 −0.5 140 SlyD- Her3 M-05- human- 32 33 4.98E−05 232 25623 0.1 20 Her3- ECD M-05- thermo −9 −6 n.d. n.d. 25 625 −0.1 20 SlyD-Her3 M-05- human- 122 123 3.74E−05 309 25 521 0.5 30 Her3- ECD M-05-thermo −3 −2 n.d. n.d. 25 525 −0.1 30 SlyD- Her3 M-05- human- 55 553.42E−05 337 25 373 0.3 44 Her3- ECD M-05- thermo −7 −6 n.d. n.d. 25 369−0.2 44 SlyD- Her3 M-05- human- 75 79 <1.00E−05  >1155 25 318 0.5 74Her3- ECD M-05- thermo 33 32 1.20E−04  96 25 315 1.1 74 SlyD- Her3 M-05-human- 0 1 n.d. n.d. 25 205 0.0 82 Her3- ECD M-05- thermo −4 −5 n.d.n.d. 25 204 −0.2 82 SlyD- Her3

It has been found that M-05-74 shows a reduced Molar Ratio in itsbinding to the human Her-3 ECD analyte (MR=0.5), whereas in its bindingto analyte Thermus thermophilus SlyD FKBP-Her3 comprising the β-hairpinHER3 (SEQ ID NO:1) M-05-74 shows an improved Molar Ratio (MR=1.1),indicating a functional, stoichiometric 1:1 binding with improvedepitope accessibility (compared to human Her-3 ECD).

b) Kinetics of HER3 Antibodies M-05-74, M-205 and M-208 Kinetics toInvestigate the Mode of Action of M-05-74 in the Absence and Presence ofHeregulin (HRG)

In its equilibrium state, the Her-3 ECD is in its “closed confirmation”,which does mean, the heterodimerization Her-3 beta-hairpin motive istethered via non-covalent interactions to the Her-3 ECD domain IV (seeFIGS. 1C and 1D). It is supposed, that the “closed” Her-3 conformationcan be opened via the binding of the ligand heregulin at a specificHer-3 heregulin binding site. This takes place at the Her-3 interfaceformed by the Her-3 ECD domains I and domain III. By this interaction itis believed, that the Her-3 receptor is activated and transferred intoits “open conformation” (see FIGS. 1B and 1E). When this occurs, theHer-3 beta-hairpin is accessible for the described antibodies. This modeof action can be simulated in vitro by a Biacore experiment.

A Biacore T100 instrument (GE Healthcare) was used to kinetically assessthe monoclonal antibodies for their behavior to the heregulin-activatedHer-3 Extracellular Domain (Her3_ECD). A CMS series sensor was mountedinto the system and was normalized in HBS-ET buffer (10 mM HEPES pH 7.4,150 mM NaCl, 3 mM EDTA, 0.005% w/v Tween 20) according to themanufacturer's instructions. The sample buffer was the system buffersupplemented with 1 mg/ml CMD (Carboxymethyldextran, Sigma #86524). Thesystem operated at 25° C. 6500 RU RAM-Fcγ (relative units ofFcγ-fragment RamIgG, GE Healthcare) were immobilized according to themanufacturer's instructions using EDC/NHS chemistry on all four flowcells. The sensor was deactivated using 1M ethanolamine.

The binding activity of the respective antibody against the analytes waskinetically tested. Antibodies were captured at 35 nM concentration by a1 min injection at 5 μl/min. The flow rate was set to 100 μl/min.

The analytes in solution tested were human Heregulin fragment (HRG) (SEQID NO:11), a 44 kDa homodimeric protein (prepared according to Example2e), human recombinant HER2 ECD (SEQ ID NO:10) (69.6 kDa), humanrecombinant HER3 ECD (SEQ ID NO:4) (68 kDa), human recombinant HER4 ECD(SEQ ID NO:6), and 100 nM of the Her-3 ECD and the Her-4 ECD eachincubated with a 5-fold molar excess of Heregulin for 60 min at roomtemperature resulting in HER3 ECD-HRG complex and HER4 ECD-HRG complex(Addition of MWs for complexes).

Analytes in solution were injected at different concentration steps of 0nM, 1.1 nM, 3.7 nM, 11.1 nM, 33.1 nM and 90 nM for 3.5 min. Thedissociation was monitored for 15 min. Where possible, kineticsignatures were evaluated according to a Langmuir fit.

TABLE 5a SPR-resolved kinetic data of M-05-74 (=M-074), M-205 and M-208CL Analyte T k_(a) k_(d) K_(D) K_(D) BL Chi² Antibody RU in solution °C. 1/Ms 1/s M nM RU MR RU² M-074 535 HRG 25 n.d. n.d. n.d. n.d. n.d.n.d. n.d. M-074 530 HER2_ECD 25 n.d. n.d. n.d. n.d. n.d. n.d. n.d. M-074648 HER3-ECD 25 1.3E+04 2.8E−05 2.2E−09 2 70 0.2 0.1 M-074 712 HER4-ECD25 6.7E+03 1.0E−03 1.5E−07 150 27 0.1 0.1 M-074 546 HER3- 25 6.3E+042.7E−04 4.2E−09 4 160 0.6 2.3 ECD-HRG M-074 719 HER4- 25 1.6E+05 8.3E−045.2E−09 5 349 0.6 0.0 ECD-HRG M-205 591 HRG 25 n.d. n.d. n.d. n.d. n.d.n.d. n.d. M-205 588 HER2_ECD 25 n.d. n.d. n.d. n.d. n.d. n.d. n.d. M-205605 HER3-ECD 25 4.9E+04 1.0E−04 2.0E−09 2 235 1.0 1.3 M-205 597 HER3- 253.7E+04 1.2E−04 3.2E−09 3 164 0.4 0.3 ECD-HRG M-208 777 HRG 25 n.d. n.d.n.d. n.d. n.d. n.d. n.d. M-208 771 HER2_ECD 25 n.d. n.d. n.d. n.d. n.d.n.d. n.d. M-208 822 HER3-ECD 25 5.8E+04 5.3E−05 9.1E−10 1 367 1.0 9.4M-208 795 HER3- 25 5.0E+04 1.4E−04 2.8E−09 3 390 1.1 17.6 ECD-HRG MR =Molar Ratio, BL = Binding Late, CL = Capture Level; n.d. = notdetectable = no binding The Molar Ratio was calculated with the formula:MW (antibody)/MW(antigen) * BL (antigen)/CL (antibody).

The antibody M-205 is a murine monoclonal antibody with binding activityversus an epitope nearby the Her-3 ECD Heregulin binding site (describedas Mab205.10.2 in WO2011/076683). M-205 competes with Heregulin aroundits binding site on the Her-3 ECD.

The antibody M-208 is a murine monoclonal antibody with binding activityversus the Her-3 ECD domain IV. M-208 binds to the Her-3 ECDindependently of the Her-3 ECD conformational state.

M-05-74 (=M-074 in Table 5) binds to the Her-3 ECD in its active “open”conformation (on the presence of ligand (e.g. heregulin HRG) withimproved kinetics, due to a better accessibility of the Her-3 hairpin inits “open” conformation. The MR is at least two fold higher.

No antibody binding (n.d.) was observed versus the negative controlanalytes Heregulin beta (HRG) and the extracellular HER-2 domain(HER2_ECD). The tested antibodies showed all binding to the Her3-ECD(HER3_ECD), but with strongly differing BL values.

M-05-74 binds to the Her-3 ECD in its “closed” conformation with slowerassociation rate constant k_(a)=1.3E+04 l/Ms and smaller BL (70 RU) thanwhen compared to the clones M-205 with faster k_(a)=4.9 E+04 l/Ms andhigh signal amplitude at BL (235 RU) and M-208 with faster k_(a)=5.8E+04l/Ms and also high signal amplitude at BL (367RU). This implicates onthe stoichiometry of the binding (MR), where M-205 (MR=1.0) and M-208(MR=1.0) both show a functional 1:1 binding for the HER3-ECD, whereasM-05-74 shows a non-functional binding (MR=0.2). Here it is supposed,that this interaction of M-05-74 versus the Her-3 ECD is residualbinding of a portion of structurally handicapped Her-3 ECD analyte. Thisis also supposed for the interaction of M-05-74 versus the Her-4 ECD,which also shows a non-functional binding with BL (27 RU) and (MR=0.1).

A surprising result is the more than 4-fold increase (nearly 5 fold) ofthe M-05-74 association rate constant k_(a) from the “closed” Her-3 ECDto the “open” Her-3 ECD/Heregulin complex; from k_(a)=1.3E+04 l/Ms(Her3_ECD) to k_(a)=6.3E+04 l/Ms (Her3-ECD-HRG). So M-05-74 binds toHER3-ECD with a ratio of the association constant (Ka) in presence ofHeregulin (Ka (+Heregulin)) and absence of Heregulin (Ka (−Heregulin))of 4.0 or higher (Ka (+Heregulin))/(Ka (−Heregulin)=ka (Her3-ECD-HRG)/ka(Her3-ECD)=6.3E+04 [l/Ms]/1.3E+04 [l/Ms])=4.85)). Thereby the MolarRatio improves 3-fold, indicating now a 1:1 interaction of M-05-74 withthe Her-3 ECD Heregulin complex. Thus binds M-05-74 to HER3-ECD with aratio of the Molar Ratio MR of binding in presence of Heregulin (MR(+Heregulin)) and in absence of Heregulin (MR (−Heregulin)) of 3.0 (MR(+Heregulin))/(MR (−Heregulin)=0.6/0.2=3).

This is also valid for the Her-4 ECD/Heregulin complex, where the MolarRatio improves 6-fold, indicating a 1:1 interaction of M-05-74 with theHer-4 ECD Heregulin complex. Thus binds M-05-74 to HER4-ECD with a ratioof the Molar Ratio MR of binding in presence of Heregulin (MR(+Heregulin)) and in absence of Heregulin (MR (−Heregulin)) of 3.0 (MR(+Heregulin))/(MR (−Heregulin)=0.6/0.1=6). And furthermore surprisinglythe M-05-74 association rate constant ka increases from the “closed”Her-4 ECD to the “open” Her-4 ECD/Heregulin complex from ka=6.7E+03 l/Ms(Her3_ECD) to ka=1.6E+05 more than 20-fold. So M-05-74 binds to HER4-ECDwith a ratio of the association constant (Ka) in presence of Heregulin(Ka (+Heregulin)) and absence of Heregulin (Ka (−Heregulin)) of 20.0 orhigher (Ka (+Heregulin))/(Ka (−Heregulin)=ka (Her4-ECD-HRG)/ka(Her4-ECD)=6.7E+04 [l/Ms]/1.6E+05 [l/Ms])=23.88)).

As expected, the Heregulin displacer M-205, reduces its BLvalue and theMolar Ratio. The Molar Ratio is decreased 2.5-fold, from a fullyfunctional 1:1 interaction with MR=1.0 (Her3-ECD) with 235 RU at BL intoa less functional MR=0.4 (Her3-ECD-HRG) with 164 RU at BL. Thisindicates the loss in functionality due to the competing presence ofexcess Heregulin.

The antibody M-208, which binds to the Her-3 ECD domain IV remainscompletely unaffected by the presence of Heregulin. No significantchange of the Molar Ratios MR could be detected.

The FIG. 7 shows the mode of binding of the anti-HER3/HER4 β-hairpinantibody M-05-74 to the Heregulin-activated Her-3 ECD complex. M-05-74(see plot 1) captures and prevents the Heregulin dissociation from thecomplex. M-05-74 is a trap for Heregulin (“Heregulin-sink”). M-05-74does not compete with Heregulin for a binding site on the Her-3 ECD. Forcomparison M-08-11 (plot 2) is shown; M-08-11 (VH and VL see SEQ ID NO:51 and 52) is another HER3 β-Hairpin binder with no HER4 ECD and HER4β-hairpin crossreactivity, which binds to a different epitope thanM-05-74.

In a further experiment also HER1 ECD, T.T.SlyD-cysHer3 and T.T.SlyD-caswithout the HER3 β-hairpin were included in the measurement—results areshown in Table 5b, which substantially reveals the same bindingproperties of M-05-74.

A Biacore T200 instrument (GE Healthcare) was mounted with a CM5 seriessensor. The sensor was normalized in HBS-ET buffer (10 mM HEPES pH 7.4,150 mM NaCl, 3 mM EDTA, 0.05% w/v Tween 20) according to themanufacturer's instructions. The sample buffer was the system buffersupplemented with 1 mg/ml CMD (Carboxymethyldextran, Sigma #86524). Thesystem operated at 25° C. 6500 RU RAM-Fcγ (relative units ofFcγ-fragment RamIgG, GE Healthcare) were immobilized according to themanufacturer's instructions using amine coupling EDC/NHS chemistry onall four flow cells. The sensor was deactivated using 1M ethanolamine.Monoclonal antibodies were captured (CL, Capture Level) on the sensorsurface by a 1 min injection at 10 μl/min. Concentration dependentkinetics were measured. A concentration series of the analytesHER-1-ECD, HER-2-ECD, HER-3-ECD, HER-4-ECD, T.T.SlyD-cysHer3 andT.T.SlyD-cas were injected each at 0 nM, 1.1 nM, 3.3 nM, 2×10 nM, 30 nMand 90 nM. Heregulin beta (HRG) was injected at 0 nM, 17 nM, 2×50 nM,150 nM and 450 nM, 90 nM HER-3 ECD and 90 nM HER-4 ECD were preincubatedfor 2 hrs with a five-fold molar excess of HRG beta and were injected atHER concentrations steps of 0 nM, 1.1 nM, 3.3 nM, 2×10 nM, 30 nM and 90nM. All analytes were injected for 5 min association time and 10 mindissociation time at 100 μl/min flow rate. The sensor capture system wasregenerated by a 3 min injection at 10 μl/min of 10 mM glycine pH 1.7.Where possible kinetic data was evaluated using the Biacore T200evaluation software. HER-3-ECD, HER-4-ECD and T.T.SlyD-cysHer3 kineticswere evaluated using a Langmuir fitting model. HER-3-ECD-HRG andHER-4-ECD-HRG kinetics of M-5-74, were evaluated according to a Langmuirfitting model.

TABLE 5b SPR-resolved kinetic data of M-05-74 CL Analyte (Ab) k_(a)k_(d) K_(D) RMax Chi² T Antibody in solution RU 1/Ms 1/s M RU MR RU² °C. M-5-74 HER1-ECD 288 n.d. n.d. n.d. 1 n.d. 0 25 HER2-ECD 287 n.d. n.d.n.d. 1 n.d. 0 HER3-ECD 289 9.6E+04 1.1E−04 1.1E−09 19 0.1 0.05 HER4-ECD285 1.6E+04 8.2E−04 5.1E−08 13 0.1 0.01 HER3- 312 1.0E+05 2.9E−042.8E−09 195 0.8 2.2 ECD-HRG HER4- 301 9.9E+04 8.1E−04 8.1E−09 179 0.81.8 ECD-HRG HRG 301 n.d. n.d. n.d. 0 n.d. 0.0 T.T.SlyD- 486 3.0E+042.4E−04 7.8E−09 88 1.9 0.02 cysHer3 T.T.SlyD- 490 n.d. n.d. n.d. 0.5 0.00.06 cas MR = Molar Ratio, BL = Binding Late, CL = Capture Level; n.d. =not detectable = no binding M-05-74 binds HER-3-ECD-HRG andHER-4-ECD-HRG with 1:1 stoichiometry and inactive HER-3-ECD andHER-4-ECD with 10:1 stoichiometry. M-05-74 binds HER-3-ECD andHER-3-ECD-HRG with higher affinity than HER-4-ECD and HER-4-ECD-HRG.M-05-74 does not interact with HER-1, HER-2 and HRG. M-05-74 bindsT.T.SlyD-cysHer3 with 1:2 stoichiometry and does not interact withT.T.SlyD-cas.

Example 4

Epitope Mapping of Anti-HER3 Antibody M-05-74 and Mode of ActionAnalysis

M-05-74 with a Unique Epitope (β-hairpin of HER3 and HER4)

A Biacore 2000 (GE Healthcare) instrument was used to assess theaccessible epitopes clone culture supernatants for their bindingspecificity. A CMS sensor was mounted into the system and was normalizedin HBS-ET buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% w/vTween 20) according to the manufacturer's instructions. The samplebuffer was the system buffer supplemented with 1 mg/ml CMD(Carboxymethyldextran, Sigma). The system operated at 37° C. 10000 RURAM-Fcγ (relative units of Fcγ-fragment Rabbit Anti-Mouse IgG/JacksonLaboratories) were immobilized according to the manufacturer'sinstructions using EDC/NHS chemistry on all four flow cells. The sensorwas deactivated using 1M ethanolamine.

At a flow rate of 10 μl/min the primary antibody 50 nM anti-HER3 M-05-74was captured for 1 min on all flow cells. The flow rate was set to 30μl/min and an IgG blocking solution (50 μg/ml IgG (20:2:1 IgG1-Fcγ,IgG2a-Fcγ, IgG2b), Roche) was injected for 5 minutes. The antigen Her-3ECD was injected at 1.5 μM for 3 min.

Afterwards, 100 nM of each anti-HER3 secondary antibodies (a) M-05-74 b)8B8 from WO97/35885 (named GT in the Figure) c) M-208 which binds todomainIV of HER3, and d) M-08-11; another HER3 β-Hairpin binder with noHER4 ECD and HER4 β-hairpin crossreactivity) was injected for 3 minutesat 30 μl/min. Acidic regeneration of the sensor surface was achievedusing three consecutive injections of 10 mM Glycine pH 1.7 at 30μl/minfor 60 sec.

The noise of the measurement is defined by the rebinding of thesecondary M-05-74 injection, which re-saturates the already dissociatedprimary M-05-74. The experiment showed (see FIG. 8), that M-208 andM-05-74 occupy distinct epitopes on the Her-3 ECD, because the secondaryM-208 signal completely saturates the Her-3 ECD in the presence ofM-05-74. M-08-11 binding is completely blocked by the presence ofM-05-74. The M-08-11 secondary signal is even below noise. NeverthelessM-08-11 binds to a different epitope than M-05-74 as M-08-11 does notbind to human HER4 ECD and HER4 β-hairpin. (see also below the exactepitope mapping data with the β-hairpins of HER3 and HER4). The 8B8(=GT) secondary antibody produces a significant signal in the presenceof M-05-74, which is above noise. Therefore the 8B8 (=GT) antibody bindsanother epitope than M-05-74 and M-08-11.

M-05-74 with Unique Epitope and Mode of Actions

A Biacore B3000 instrument (GE Healthcare) was used to kineticallyassess the clone culture M-05-74 and the antibody 8B8 (from WO 97/35885,named GT in the Figures) to the “closed” conformation of Her-3 ECD andthe “open”, Heregulin-activated Her-3 ECD. A CMS series sensor wasmounted into the system and was normalized in HBS-ET buffer (10 mM HEPESpH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% w/v Tween 20) according to themanufacturer's instructions. The sample buffer was the system buffersupplemented with 1 mg/ml CMD (Carboxymethyldextran). The systemoperated at 25° C. 10000 RU RAM-Fcγ (relative units of Fcγ-fragmentRabbit Anti-Mouse IgG/Jackson Laboratories) were immobilized accordingto the manufacturer's instructions using EDC/NHS chemistry on all flowcells. The sensor was deactivated using 1M ethanolamine. Analytes insolution were injected at 100 μl/min at different concentration steps of0 nM, 1.1 nM, 3.7 nM, 11.1 nM, 33.1 nM and 90 nM for 2 min. Thedissociation was monitored for 5 min. Acidic regeneration of the sensorsurface was achieved using three consecutive injections of 10 mM GlycinepH 1.7 at 30 μl/min for 60 sec. Kinetic data were evaluated according toa Langmuir fit.

TABLE 6 Langmuir kinetics of M-05-74 in comparison to 8B8 (GT). 8B8 withlower antigen complex stability (t/2diss) and less functionality (MR).CL Analyte in T t/2-diss Chi² Antibody (RU) solution (° C.) ka (1/Ms)(min) BL (RU) MR (RU²) 8B8 339.3 ECD-HRG 25 3.21E+05 0.8 90 0.4 2.57M-074 314.7 ECD-HRG 25  6.6E+04 18 199 0.8 0.773 8B8 347.3 Her-3 ECD 251.02E+05 5.3 13.1 0.1 0.12 M-074 318.2 Her-3 ECD 25 2.04E+04 28 36 0.20.122 8B8 476.1 ttSlyD-Her3 25 n.d. n.d. n.d. n.d. n.d. M-074 468ttSlyD-Her3 25 8.75E+04 4.9 68.1 1.5 0.174 MR = Molar Ratio, BL =Binding Late, CL = Capture Level

In the table above kinetic data of the antibody clone M-05-74 and theantibody 8B8 are listed. M-05-74 binds to the Heregulin-activated Her-3ECD with high functionality MR=0.8. M-05-74 and acts as Heregulin trap.(see also Figure Biacore sensogram Example 3b and FIG. 7).

The complex stability of the 8B8 antibody with t1/2 diss=0.8 min isweak. 8B8 binds with an, MR=0.4.No separated dissociation phases of the8B8 antibody and the Heregulin dissociation can be identified. Heregulincompletely dissociates off in the same timeframe and with the samevelocity, like 8B8. 8B8 antibody does not delay the heregulindissociation.

M-05-74 functionally binds (MR=1.5) to the Thermus thermophilus SlyDFKBP-Her3 comprising th HER3 β-Hairpin of SEQ ID NO:1 with KD=27 nM.Since the antibody 8B8 does not bind to the HER3 β-Hairpin comprisingThermus thermophilus SlyD FKBP-Her-3 fusion polypeptide this antibodytargets another epitope than M-05-74.

FIG. 9 is an overlay plot of the biacore sensogramms of anti-HER3/HER4antibody M-05-74, anti-HER3 antibody M-08-11 and anti-HER3 antibody 8B8(from WO97/35885) showing the different binding modes of actions.Anti-HER3/HER4 antibody M-05-74 traps the Heregulin-activated Her-3 ECD(1) with t1/2 diss=18 min and acts Heregulin-sink. Anti-HER3 antibodyM-08-11 HER3 (β-Hairpin binder with no HER4 ECD and HER4 β-hairpincrossreactivity) delays the Heregulin dissociation (2) and produces acomplex two-state kinetic. 8B8 antibody (3) is does not trap Heregulinand also not delays the Heregulin dissociation from the Her-3ECD/Heregulin complex. Since it is a perfect Langmuir interaction, theHeregulin/Her-3 ECD complex quickly and completely dissociates as intactcomplex from the 8B8 antibody.

In FIG. 10 a scheme of these binding modes of action is shown: 1:M-08-11 binds to the Heregulin activated Her-3 ECD and induces a delayedHeregulin dissociation, whereby M-08-11 stays in the Her-3 ECD receptorcomplex. 2: M-05-74 binds to the Heregulin activated Her-3 ECD.Heregulin is trapped in the complex and the antibody stays in thecomplex. 3: 8B8 binds the Heregulin activated Her-3 ECD. The wholecomplex dissociates from the antibody.

Peptide-Based 2D Epitope Mapping

In another embodiment a peptide-based epitope mapping experiment wasdone to characterize the Her-3 ECD epitopes by using the CelluSpots™Synthesis and Epitope Mapping technology. Epitope mappings were carriedout by means of a library of overlapping, immobilized peptide fragments(length: 15 amino acids) corresponding to the sequences of human Her-1ECD, Her-2 ECD, Her-3 ECD and Her-4 ECD peptide hairpins. In FIG. 11,the strategy of the epitope mapping and alanine-scan approach is shown.The peptide hairpin sequences (β-hairpin) of HER1(EGFR) ECD, HER2 ECD,HER3 ECD and HER4 ECD including their structural embeddings (structural)were investigated. Cysteins were replaced by serines. For antibodyselection of the antibodies via binding to such β-hairpins, theβ-hairpins of HER3 and HER4 are defined by SEQ ID NO:1 and SEQ ID NO:2.

Each peptide synthesized was shifted by one amino acid, i.e. it had 14amino acids overlap with the previous and the following peptide,respectively. For preparation of the peptide arrays the IntavisCelluSpots™ technology was employed. In this approach, peptides aresynthesized with an automated synthesizer (Intavis MultiPep RS) onmodified cellulose disks which are dissolved after synthesis. Thesolutions of individual peptides covalently linked to macromolecularcellulose are then spotted onto coated microscope slides, TheCelluSpots™ synthesis was carried out stepwise utilizing9-fluorenylmethoxycarbonyl (Fmoc) chemistry on amino-modified cellulosedisks in a 384-well synthesis plate. In each coupling cycle, thecorresponding amino acids were activated with a solution of DIC/HOBt inDMF. Between coupling steps un-reacted amino groups were capped with amixture of acetic anhydride, diisopropylethyl amine and1-hydroxybenzotriazole. Upon completion of the synthesis, the cellulosedisks were transferred to a 96-well plate and treated with a mixture oftrifluoroacetic acid (TFA), dichloromethane, triisoproylsilane (TIS) andwater for side chain deprotection. After removal of the cleavagesolution, the cellulose bound peptides are dissolved with a mixture ofTFA, TFMSA, TIS and water, precipitated with diisopropyl ether andre-suspended in DMSO. The peptide solutions were subsequently spottedonto Intavis CelluSpots™ slides using an Intavis slide spotting robot.

For epitope analysis, the slides prepared as described above were washedwith ethanol and then with Tris-buffered saline (TBS; 50 mM Tris, 137 mMNaCl, 2.7 mM KCl, pH 8) before blocking for 16 h at 4° C. with 5 mL 10×Western Blocking Reagent (Roche Applied Science), 2.5 g sucrose in TBS,0.1% Tween 20. The slide was washed with TBS and 0.1% Tween 20 andincubated afterward with 1 μg/mL of the corresponding IGF1 antibodies inTBS and 0.1% Tween 20 at ambient temperature for 2 h and subsequentlywashed with TBS +0.1% Tween 20. For detection, the slide was incubatedwith anti-rabbit/anti-mouse secondary HRP-antibody (1:20000 in TBS-T)followed by incubation with chemiluminescence substrate luminol andvisualized with a Lumilmager (Roche Applied Science). ELISA-positiveSPOTs were quantified and through assignment of the correspondingpeptide sequences the antibody binding epitopes were identified.

As depicted in FIG. 12, M-05-74 shows a HER3 ECD epitope with the aminoacid sequence VYNKLTFQLEP (SEQ ID NO:43) and a crossreactivity to a HER4ECD epitope with the amino acid sequence VYNPTTFQLE (SEQ ID NO:44) withno detectable signals versus the hairpin motives in EGFR and the HER2ECD. No signals at all were detectable with the 8B8 antibody, thereforethe 8B8 antibody targets epitopes, different from the hairpin peptidemotives. M-08-11 shows a HER3 ECD specific epitope with the amino acidsequence PLVYNKLTFQLE with no crossreactivity detectable to the otherhairpin sequences of the Her-family.

In FIG. 13, the amino acids identified by Ala-Scan which arecontributing most to the binding of antiHER3/HER4 antibody M-05-74 toits HER3 ECD binding epitope VYNKLTFQLEP (SEQ ID NO:43) and to its HER4ECD binding epitope VYNPTTFQLE (SEQ ID NO:44) are underlined/bold.

Example 5

Binding of HRG to HER3-ECD in the Presence of HER3 Antibody (ELISA)

A Streptavidin-coated 96-well plate was incubated at 4° C. with cellculture supernatant containing SBP-tagged HER3-ECD. On the next day thewells were washed three times with washing buffer (PBS+0.05% Tween-20)and blocked with PBS containing 1% BSA for one hour. After another threewashes with washing buffer, 40 μl antibody solution (in Delfia BindingBuffer) was added to each well as a 2× stock of the desired finalconcentrations (10⁻³ to 10³ nM, alternatively 10⁻⁴ to 10² nM).Immediately 40 μl of 20 nM Europium-labeled Heregulin-beta (PeproTech,Cat. #100-03) was added to achieve a final concentration of 10 nM. Theplates were incubated on a shaker at room temperature for two hours.Following three washes with Delfia Wash Buffer, Delfia EnhancementSolution was added and incubated on a shaker for 15 minutes (lightprotected). Finally, the plates were measured in a Tecan Infinite F200reader using a time-resolved fluorescence measurement protocol. Thebinding of M-05-74 (named M-074 in FIG. 14) can promote binding of HRGto HER3-ECD until a plateau is reached at a signal of 650. Results areshown in FIG. 14.

Example 6

a) Inhibition of HER3 Phosphorylation in ZR-75-1 Cells

Assays were performed in ZR-75-1 cells according to the followingprotocol: Seed cells with 500,000 cells/well into Poly-D-Lysine coated6-well plate in RPMI1640 medium with 10% FCS. Incubate for 24 h. Removemedium by aspirating, incubate overnight with 500 μl/well RPMI 1640 with0.5% FCS. Add antibodies in 500 μl RPMI 1640 with 0.5% FCS. Incubate forlh. Add Heregulin-beta (PeproTech, Cat. #100-03)) (final concentration500 ng/ml) for 10 min. To lyse the cells remove medium and add 80 μl icecold Triton-X-100 cell lysis buffer and incubate for 5 minutes on ice.After transferring the lysate into 1.5 ml reaction tube andcentrifugation at 14000 rpm for 15 min at 4° C., transfer supernatantinto fresh reaction tubes. Samples containing equal amounts of proteinin SDS loading buffer were separated on SDS PAGE and blotted by using asemi-dry Western Blot to nitrocellulose membranes. Membrans were blockedby 1× NET-buffer+0.25% gelatine for 1 h hour and pHER3 is detected bythe antibody αPhospho-HER3/ErbB3 (Tyr1289) (21D3), Cell Signaling,#4791and HER3 by the antibody αErbB3 (C-17), Santa Cruz, #sc-285respectively. After washing and detection of the signals by an PODcoupled secondary antibody, bands were densometricaly scanned. Percent(%) inhibition of anti-HER3 antibodies M-05-74 on receptorphosphorylation in zr-75-1 cells is shown below in Table 7.

TABLE 7 % Inhibition of HER3 phosphorylation in ZR-75-1 cells pHER3 %inhibiton antibody [10 μg/ml] Ctrl 0 M-05-74 49b) Inhibition of HER3 Phosphorylation of the Bivalent Parent M-05-74 andthe Fab Fragment of M-05-74 (Fab-74)

MCF-7 cells were seeded into 24-Well-plates (1 ml RPMI, 10% FCS, 3×105cells per well) and were incubated at 37° C./5% CO2 overnight. After 24hours the media was replaced with 1 ml media containing 0.5% FCS. After48 hours the antibodies were added to a final concentration of 10 μg/ml,1 μg/ml and 0.1 μg/ml (M-05-74) and 6.66 μg/ml, 0.66 μg/ml and 0.066μg/ml (Fab-074). The plates were incubated at 37° C. for 50 minutes andthen Heregulin-beta (PeproTech, Cat. #100-03) was added to a finalconcentration of 500 ng/ml. The plates were incubated for a further 10minutes at 37° C./5% CO2. The cells were washed with PBS and lysed in 40μl Triton Lysis Buffer (1% Triton) containing Aprotinin (10 μg/ml),Orthovanadate (0.4 mM), Phenylmethylsulfonyl fluoride (1 mM). 26 μl ofthe collected lysates were transferred to reaction tubes and 14 μlSample Buffer (NuPAGE LDS Sample Buffer 4×, NuPAGE Sample Reducing Agent10×) was added. The samples were incubated for 10 minutes at 70° C. andthen analysed by SDS-PAGE (NuPAGE, 4-12% Bis-Tris-Mini-Gel).Electroblotting was performed using the iBlot Dry Blotting System(Invitrogen). The nitrocellulose membrane was incubated with phosphoHER3antibody (α Phospho Her3, Cellsignaling #4791, Rabbit 1:1000) followedby incubation with HRP-conjugated secondary antibody (goat anti rabbit1:5000, BioRad cat: 170-6515). Signal was developed using ECL DetectionReagents (Amersham RPN2209) on X-Ray film (Roche Lumi-FilmChemiluminescent Detection Film 11666657001). The anti-HER3 antibodyM-05-74 (full length purified from hybridoma) and the Fab fragment ofthe antibody Fab-74 (obtained py papain cleavage from full lengthM-05-74) were investigated in eqimolar amounts. Fab fragments weregenerated by papain digestion of the antibody. Briefly, 1 ml of app. 2mg/ml antibody containing solution was supplemented with 25 mM Cysteinand 70 μg papain (Roche). After incubation at 37° C. for 1.5 h, thedigestion reaction was stopped by addition of iodoacetamide and thereaction mixture was purified by Mab Select Sure (GE Healthcare). TheFab containing flowthrough fraction was further purified by sizeexclusion chromatography (Superdex 200; GE Healthcare).

Percent (%) inhibition of anti-HER3 antibodies on receptorphosphorylation in MCF7 cells is summarised below and in Table 8. Theantibody M-05-74 (full length from hybridoma) and the Fab fragment ofthis antibody Fab-74 can inhibit HER3 phosporylation in equimolarconcentrations to an comparable extent.

TABLE 8 % Inhibition of HER3 phosphorylation in MCF-7 cells pHER3 pHER3% inhibition % inhibition Antibody [6.66 nM] [0.66 nM] control 0 0M-05-74 94 13 (full length from hybridoma) Fab 96 14 fragment of M-05-74(Fab-74)

Example 7

Inhibition of HER2/HER3 Heterodimers (Imunoprecipitation and WesternBlot) in MCF7 Cells

MCF-7 cells were seeded into 6-Well-plates (2 ml RPMI, 10% FCS, 8×105cells per well) and were grown overnight. On the next day the media wasexchanged by 2 ml starving media containing 0.5% FCS. On day three theantibodies were added to a final concentration of 10 μg/ml and theplates were incubated at 37° C. After 50 minutes Heregulin-beta(PeproTech, Cat. #100-03) was added to a final concentration of 500ng/ml and the plates were incubated for another 10 minutes at 37° C. Thecells were washed with PBS and lysed in 250 μl Triton Lysis Buffercontaining 1% Digitonin. 60 μl of the collected lysates were transferredto reaction tubes and incubated with 40 μl antibody-coupled Sepharose(either Herceptin or HER3-antibody #208) and 500 μl Buffer containing0.3% Digitonin. The reaction mixes were incubated on a wheel rotatorovernight at 4° C. On the next day the reaction mixes were washed threetimes with 500 μl Buffer containing 0.3% Digitonin. After the last washthe supernatant was discarded and 10 μl 4× Loading Buffer was added. Thetubes were incubated for 10 minutes at 70° C. and the supernatants wereconsequently loaded onto a gel for SDS-PAGE. After the followingSemi-Dry Western Blot the membranes containing the samplesimmunoprecipitated with HER2 antibody were incubated with anti-HER3/HER4antibody M-05-74 (M-074 in FIG. 15), and vice versa. The membranes werethen incubated with HRP-conjugated secondary antibody and the ECL signalwas transferred onto X-Ray film. Results are shown in FIG. 15, showing astrong inhibition of the HER2/HER heterodimer formation (HER2/HERheterodimerization) by the M-05-74.

Example 8

Inhibition of Tumor Cell Proliferation of M-05-74 in MDA-MB-175 Cells.

The anti-tumor efficacy of HER3 antibodies M-05-74 in a cellproliferation assay, using MDA-MB-175 cells (VII Human Breast CarcinomaCells, ATCC catalog no. HTB-25), was assessed. 20,000 cells per wellwere seeded into sterile 96 well tissue culture plates with DMEM/F12cell culture medium, containing 10% FCS and incubated at 37° C.±1° C.with 5%±1% CO₂ for one day. The cells are slow growing cells with adoubling time of ca. 3 days. Anti-HER3 antibodies were added in dilutionseries and further incubated for 6 days. Cell viability was thenassessed using the alamarBlue® readout. EC50 values were calculated.

TABLE 9 EC50 of the Inhibition of tumor cell proliferation of M-05-74 inMDA- MB-175 cells antibody EC₅₀ [μg/ml] M-05-74 5.8

Example 9

In Vivo Antitumor Efficacy of Anti-HER3 Antibody M-05-74

The in vivo antitumor efficacy of the anti-HER3 antibody M-05-74 (M-074)could be detected in cell based models of various tumor origin (e.g.SCCHN and pancreatic cancer) transplanted on SCID beige. As example dataare shown for the SCCHN xenograft model FaDu (cell line based).

Test Agents

M-05-74 was provided as stock solution from Roche, Penzberg, Germanyexpressed and purified from hybridoma cells. Antibody buffer includedhistidine. Antibody solution was diluted appropriately in buffer fromstock prior injections.

Cell Lines and Culture Conditions

FaDu human HNSCC cells were originally obtained from ATCC. The tumorcell line was routinely cultured in MEM Eagle medium supplemented with10% fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate and 0.1mM NEAA at 37° C. in a water-saturated atmosphere at 5% CO2. Culturepassage was performed with trypsin/EDTA 1× splitting every third day.

Animals

Female SCID beige or nude mice were purchased from breeder (e.g. CharlesRiver, Sulzfeld, Germany) and maintained under specific-pathogen-freecondition with daily cycles of 12 h light/12 h darkness according tocommitted guidelines (GV-Solas; Felasa; TierschG). Experimental studyprotocol was reviewed and approved by local government. After arrivalanimals were maintained in the quarantine part of the animal facilityfor one week to get accustomed to new environment and for observation.Continuous health monitoring was carried out on regular basis. Diet food(Provimi Kliba 3337) and water (acidified pH 2.5-3) were provided adlibitum.

Animals were controlled daily for clinical symptoms and detection ofadverse effects. For monitoring throughout the experiment body weight ofanimals was documented.

Animal treatment started after animal randomisation after celltransplantation when median tumor size was about 100-150 mm3. Antibodywas administered as single agent at 10 mg/kg i.p. q7d once weekly forseveral weeks depending of the model. The corresponding vehicle wasadministered on the same days.

FaDu HNSCC xenograft bearing mice were treated with antibody M-05-74from study day 10 to 24. As a result, treatment with H-74 antibodyshowed significant anti-tumor efficacy with nearly tumors stasis of s.c.FaDu xenografts. The Tumor Growth Inhibition (TGI) was calculated at89%.

Treatment with M-05-74 (10 mg/kg q7dx3, i.p.) resulted in nearly tumorstasis of FaDu. Results are shown in FIG. 17, wherein M-05-74 is namedM-074.

Example 10

Generation of M-05-74-Fab-Pseudomonas Exotoxin Conjugate (M-05-74-PE)

Expression, purification and renaturation of Fab fragment of M-05-74,PE24 variant, and Fab fragment of M-05-74 conjugated to Pseudomonasexotoxin variant PE24LR8M based on the Sequences of SEQ ID NO:45, 46,47, 48 (or 49).

Expression of Fab (e.g. for Sortase Coupling)-Expression Vectors

For the expression of the described Fab fragments, variants ofexpression plasmids for transient expression (e.g. HEK293-F) cells basedeither on a cDNA organization with or without a CMV-Intron A promoter oron a genomic organization with a CMV promoter were applied.

Beside the antibody expression cassette the vectors contained:

-   -   an origin of replication which allows replication of this        plasmid in E. coli, and    -   a β-lactamase gene which confers ampicillin resistance in E.        coli.

The transcription unit of the antibody gene was composed of thefollowing elements:

-   -   unique restriction site(s) at the 5′ end    -   the immediate early enhancer and promoter from the human        cytomegalovirus,    -   followed by the Intron A sequence in the case of the cDNA        organization,    -   a 5′-untranslated region of a human antibody gene,    -   an immunoglobulin heavy chain signal sequence,    -   the human antibody chain either as cDNA or as genomic        organization with the immunoglobulin exon-intron organization    -   a 3′ untranslated region with a polyadenylation signal sequence,        and    -   unique restriction site(s) at the 3′ end.

The fusion genes comprising the antibody chains as described below weregenerated by PCR and/or gene synthesis and assembled by knownrecombinant methods and techniques by connection of the accordingnucleic acid segments e.g. using unique restriction sites in therespective vectors. The subcloned nucleic acid sequences were verifiedby DNA sequencing. For transient transfections larger quantities of theplasmids were prepared by plasmid preparation from transformed E. colicultures (Nucleobond AX, Macherey-Nagel).

Cell Culture Techniques

Standard cell culture techniques were used as described in CurrentProtocols in Cell Biology (2000), Bonifacino, J. S., Dasso, M., Harford,J. B., Lippincott-Schwartz, J. and Yamada, K. M. (eds.), John Wiley &Sons, Inc.

The Fab fragments were expressed by transient co-transfection of theexpression plasmids of the heavy and the light chain in HEK29-F cellsgrowing in suspension as described below.

Transient Transfections in HEK293-F System

The Fab fragments were generated by transient transfection with therespective plasmids (e.g. encoding the heavy and modified heavy chain,as well as the corresponding light and modified light chain) using theHEK293-F system (Invitrogen) according to the manufacturer'sinstruction. Briefly, HEK293-F cells (Invitrogen) growing in suspensioneither in a shake flask or in a stirred fermenter in serum-freeFreeStyle™ 293 expression medium (Invitrogen) were transfected with amix of the four expression plasmids and 293-Free™ (Novagen) or Fectin(Invitrogen). For 2 L shake flask (Corning) HEK293-F cells were seededat a density of 1.0E*6 cells/mL in 600 mL and incubated at 120 rpm, 8%CO2. The day after the cells were transfected at a cell density of ca.1.5E*6 cells/mL with ca. 42 mL mix of A) 20 mL Opti-MEM (Invitrogen)with 600 μg total plasmid DNA (1 μg/mL) encoding the heavy or modifiedheavy chain, respectively and the corresponding light chain in anequimolar ratio and B) 20 ml Opti-MEM+1.2 mL 293-Free (Novagen) orFectin (2 μl/mL). According to the glucose consumption glucose solutionwas added during the course of the fermentation. The supernatantcontaining the secreted antibody was harvested after 5-10 days andantibodies were either directly purified from the supernatant or thesupernatant was frozen and stored.

Expression of Pseudomonas Exotoxin Variant PE24-LR8M for SortaseCoupling-Expression Vector

For the expression of PE24-LR8M an E. coli expression plasmid was used.

Beside the expression cassette for the pseudomonas exotoxin A domain IIIthe vector contained:

-   -   an origin of replication from the vector pBR322 for replication        in E. coli (according to Sutcliffe, G., et al., Quant. Biol.        43 (1979) 77-90),    -   the lacI repressor gene from E. coli (Farabaugh, P. J., Nature        274 (1978) 765-769),    -   the URA3 gene of Saccharomyces cerevisiae coding for orotidine        5′-phosphate decarboxylase (Rose, M. et al. Gene 29 (1984)        113-124) which allows plasmid selection by complementation of E.        coli pyrF deletion strains (uracil auxotrophy).

The transcription unit of the toxin gene was composed of the followingelements:

-   -   unique restriction site(s) at the 5′ end,    -   the T5 hybrid promoter (T5-PN25/03/04 hybrid promoter according        to Bujard, H., et al. Methods. Enzymol. 155 (1987) 416-433 and        Stueber, D., et al., Immunol. Methods IV (1990) 121-152)        including a synthetic ribosomal binding site according to        Stueber, D., et al. (see before),    -   the pseudomonas exotoxin A domainIII with an N-terminal coupling        tag followed by a furin site (SEQ ID NO:45 Pseudomonas exotoxin        variant PE24LR8M_3G, including a GGG linker for sortase        coupling),    -   two bacteriophage-derived transcription terminators, the λ-T0        terminator (Schwarz, E., et al., Nature 272 (1978) 410-414) and        the fd-terminator (Beck E. and Zink, B. Gene 1-3 (1981) 35-58),    -   unique restriction site(s) at the 3′ end.        Cultivation and Expression of the Pseudomonas Exotoxin A        Construct Variant PE24-LR8M _3G in an E. coli Fed-batch Process        on Chemical Defined Medium

For the expression of PE24-LR8M_3G_E. coli (25 kDa) the E. colihost/vector system which enables an antibiotic-free plasmid selection bycomplementation of an E. coli auxotrophy (PyrF) was employed (EP 0 972838 and U.S. Pat. No. 6,291,245).

An E. coli K12 strain was transformed by electroporation with theexpression plasmid. The transformed E. coli cells were first grown at37° C. on agar plates. A colony picked from this plate was transferredto a 3mL roller culture and grown at 37° C. to an optical density of 1-2(measured at 578 nm). Then 1000 μl culture where mixed with 1000 μlsterile 86%-glycerol and immediately frozen at −80° C. for long timestorage. The correct product expression of this clone was first verifiedin small scale shake flask experiments and analyzed with SDS-Page priorto the transfer to the 10 L fermenter.

Pre Cultivation:

For pre-fermentation a chemical defined medium has been used. Forpre-fermentation 220 ml of medium in a 1000 ml Erlenmeyer-flask withfour baffles was inoculated with 1.0 ml out of a primary seed bankampoule. The cultivation was performed on a rotary shaker for 8 hours at32° C. and 170 rpm until an optical density (578 nm) of 2.9 wasobtained. 100 ml of the pre cultivation was used to inoculate the batchmedium of the 10L bioreactor.

Fermentation:

For fermentation in a 101 Biostat C, DCU3 fermenter (Sartorius,Melsungen, Germany) a chemical defined batch medium was used. Allcomponents were dissolved in deionized water. The alkaline solution forpH regulation was an aqueous 12.5% (w/v) NH₃ solution supplemented with11.25 g/l L-methionine.

Starting with 4.2 1 sterile batch medium plus 100 ml inoculum from thepre cultivation the batch fermentation was performed at 31° C., pH6.9±0.2, 800 mbar back pressure and an initial aeration rate of 10l/min. The relative value of dissolved oxygen (pO2) was kept at 50%throughout the fermentation by increasing the stirrer speed up to 1500rpm. After the initially supplemented glucose was depleted, indicated bya steep increase in dissolved oxygen values, the temperature was shiftedto 25° C. and 15 minutes later the fermentation entered the fed-batchmode with the start of both feeds (60 and 14 g/h respectively). The rateof feed 2 is kept constant, while the rate of feed 1 is increasedstepwise with a predefined feeding profile from 60 to finally 160 g/hwithin 7 hours. When carbon dioxide off gas concentration leveled above2% the aeration rate was constantly increased from 10 to 20 l/min within5 hours. The expression of recombinant PE24-LR8M_3G_E. coli protein wasinduced by the addition of 2.4 g IPTG at an optical density of approx.120. The target protein is expressed soluble within the cytoplasm.

After 24 hours of cultivation an optical density of 209 is achieved andthe whole broth is cooled down to 4-8° C. The bacteria are harvested viacentrifugation with a flow-through centrifuge (13,000 rpm, 13 l/h) andthe obtained biomass is stored at −20° C. until further processing (celldisruption). The yield is 67.5 g dry cells per liter.

Analysis of Product Formation:

Samples drawn from the fermenter, one prior to induction and the othersat dedicated time points after induction of protein expression areanalyzed with SDS-Polyacrylamide gel electrophoresis. From every samplethe same amount of cells (OD_(Target)=10) are suspended in 5 mL PBSbuffer and disrupted via sonication on ice. Then 100 μL of eachsuspension are centrifuged (15,000 rpm, 5 minutes) and each supernatantis withdrawn and transferred to a separate vial. This is to discriminatebetween soluble and insoluble expressed target protein. To eachsupernatant (=soluble protein fraction) 100 μL and to each pellet(=insoluble protein fraction) 200 μL of SDS sample buffer (Laemmli, U.K., Nature 227 (1970) 680-685) are added. Samples are heated for 15minutes at 95° C. under intense mixing to solubilize and reduce allproteins in the samples. After cooling to room temperature 5 μL of eachsample are transferred to a 4-20% TGX Criterion Stain Freepolyacrylamide gel (Bio-Rad). Additionally 5 μl molecular weightstandard (Precision Plus Protein Standard, Bio-Rad) were applied.

The electrophoresis was run for 60 Minutes at 200 V and thereafter thegel was transferred the GelDOC EZ Imager (Bio-Rad) and processed for 5minutes with UV radiation. Gel images were analyzed using Image Labanalysis software (Bio-Rad). Relative quantification of proteinexpression was done by comparing the volume of the product bands to thevolume of the 25 kDa band of the molecular weight standard.

Cultivation and Expression of an Antibody Fragment Light Chain Construct(VL) and an Antibody Fragment Heavy Chain Pseudomonas Exotoxin A VariantFusion (Fab-PE24) in an E. coli Fed-batch Process on Chemical DefinedMedium

For the expression of a Fab-light chain (23.4 kDa) and a Fab-heavy chainPE24 fusion (48.7 kDa) the E. coli host/vector system which enables anantibiotic-free plasmid selection by complementation of an E. coliauxotrophy (PyrF) was employed (EP 0 972 838 and U.S. Pat. No.6,291,245).

An E. coli K12 strain was transformed by electroporation with therespective expression plasmids. The transformed E. coli cells were firstgrown at 37° C. on agar plates. For each transformation a colony pickedfrom this plate was transferred to a 3 mL roller culture and grown at37° C. to an optical density of 1-2 (measured at 578 nm). Then 1000 μlculture where mixed with 1000 μl sterile 86%-glycerol and immediatelyfrozen at −80° C. for long time storage. The correct product expressionof these clones was first verified in small scale shake flaskexperiments and analyzed with SDS-Page prior to the transfer to the 10 Lfermenter.

Pre-cultivation:

For pre-fermentation a chemical defined medium has been used. Forpre-fermentation 220 ml of medium in a 1000 ml Erlenmeyer-flask withfour baffles was inoculated with 1.0 ml out of a primary seed bankampoule. The cultivation was performed on a rotary shaker for 9 hours at37° C. and 170 rpm until an optical density (578 nm) of 7 to 8 wasobtained. 100 ml of the pre cultivation was used to inoculate the batchmedium of the 10 L bioreactor.

Fermentation (RC52 #003):

For fermentation in a 101 Biostat C, DCU3 fermenter (Sartorius,Melsungen, Germany) a chemical defined batch medium was used. Thealkaline solution for pH regulation was an aqueous 12.5% (w/v) NH₃solution supplemented with 11.25 g/l L-methionine.

Starting with 4.2 l sterile batch medium plus 100 ml inoculum from thepre cultivation the batch fermentation was performed at 31° C., pH6.9±0.2, 800 mbar back pressure and an initial aeration rate of 10l/min. The relative value of dissolved oxygen (pO2) was kept at 50%throughout the fermentation by increasing the stirrer speed up to 1500rpm. After the initially supplemented glucose was depleted, indicated bya steep increase in dissolved oxygen values, the temperature was shiftedto 37° C. and 15 minutes later the fermentation entered the fed-batchmode with the start of both feeds (60 and 14 g/h respectively). The rateof feed 2 is kept constant, while the rate of feed 1 is increasedstepwise with a predefined feeding profile from 60 to finally 160 g/hwithin 7 hours. When carbon dioxide off gas concentration leveled above2% the aeration rate was constantly increased from 10 to 20 l/min within5 hours. The expression of recombinant target proteins as insolubleinclusion bodies located in the cytoplasm was induced by the addition of2.4 g IPTG at an optical density of approx. 40.

After 24 hours of cultivation an optical density of 185 is achieved andthe whole broth is cooled down to 4-8° C. The bacteria are harvested viacentrifugation with a flow-through centrifuge (13,000 rpm, 13 l/h) andthe obtained biomass is stored at −20° C. until further processing (celldisruption). The yield is between 40 and 60 g dry cells per liter.

Analysis of Product Formation:

Samples drawn from the fermenter, one prior to induction and the othersat dedicated time points after induction of protein expression areanalyzed with SDS-Polyacrylamide gel electrophoresis. From every samplethe same amount of cells (OD_(Target)=10) are suspended in 5 mL PBSbuffer and disrupted via sonication on ice. Then 100 μL of eachsuspension are centrifuged (15,000 rpm, 5 minutes) and each supernatantis withdrawn and transferred to a separate vial. This is to discriminatebetween soluble and insoluble expressed target protein. To eachsupernatant (=soluble protein fraction) 100 μL and to each pellet(=insoluble protein fraction) 200 μL of SDS sample buffer (Laemmli, U.K., Nature 227 (1970) 680-685) are added. Samples are heated for 15minutes at 95° C. under intense mixing to solubilize and reduce allproteins in the samples. After cooling to room temperature 5 μL of eachsample are transferred to a 4-20% TGX Criterion Stain Freepolyacrylamide gel (Bio-Rad). Additionally 5 μl molecular weightstandard (Precision Plus Protein Standard, Bio-Rad) and 3 amounts (0.3μl, 0.6 μl and 0.9 μl) quantification standard with known target proteinconcentration (0.1 μg/μl) were applied.

The electrophoresis was run for 60 Minutes at 200 V and thereafter thegel was transferred the GelDOC EZ Imager (Bio-Rad) and processed for 5minutes with UV radiation. Gel images were analyzed using Image Labanalysis software (Bio-Rad). With the three standards a linearregression curve was calculated with a coefficient of >0.99 and thereofthe concentrations of target protein in the original sample wascalculated.

Purification, Sortase Coupling and Renaturation (of Fab Fragment ofM-05-74, PE24 Variant, and Fab Fragment of M-05-74 Conjugated toPseudomonas Exotoxin Variant PE24LR8M)

Fab Fragment

The Fab fragment was purified by affinity chromatography (Ni Sepharose™High Perfomance HisTrap™) according to the manufacture's description. Inbrief, the supernatant was loaded onto the column equilibrated in 50 mMsodium phosphate pH 8.0, 300 mM NaCl. Protein elution was performed withthe same buffer at pH 7.0 with a washing step containing 4 mM imidazolefollowed by a gradient up to 100 mM imidazole. Fractions containing thedesired Fab fragment were pooled and dialyzed against 20 mM His, 140 mMNaCl, pH 6.0.

PE24 for Sortase Coupling

E. coli cells expressing PE24 were lysed by high pressure homogenization(if details are required: Christian Schantz) in 20 mM Tris, 2 mM EDTA,pH 8.0+Complete protease inhibitor cocktail tablets (Roche). The lysatewas filtrated and loaded onto a Q sepharose FF (GE Healthcare)equilibrated in 20 mM Tris, pH 7.4. Protein was eluted with a gradientup to 500 mM NaCl in the same buffer. PE24 containing fractions wereidentified by SDS PAGE. The combined pool was concentrated and appliedto a HiLoad™ Superdex™ 75 (GE Healthcare) equilibrated in 20 mM Tris,150 mM NaCl, pH 7.4. Fractions containing PE24 were pooled according toSDS PAGE and frozen at −80° C.

Sortase Coupling of Fab Fragment to PE24

Fab fragment and PE24 were diafiltrated separately into 50 mM Tris, 150mM NaCl, 5 mM CaCl₂ pH7.5 using Amicon® Ultra 4 centrifugal filterdevices (Merck Millipore) and concentrated to 5-10 mg/ml. Both proteinsand sortase were combined in a 1:1:0.8 molar ratio. After one hourincubation at 37° C. the mixture was loaded onto a Ni Sepharose™ HighPerfomance HisTrap™) equilibrated in 50 mM sodium phosphate, pH 8.0, 300mM NaCl. Elution was performed with a gradient up to 100 mM imidazole inthe same buffer pH 7.0. The flow through fractions containing the finalproduct Fab-PE24 was concentrated and loaded onto a HiLoad™ Superdex™200 (GE Healthcare) in 20 mM Tris, 150 mM NaCl, pH 7.4. Fractionscontaining the desired coupled protein were pooled and stored at −80° C.As sortase soluble S. aureus sortase A was used (SEQ ID NO: 50). SolubleS. aureus sortase A was expressed and purified using the followingexpression plasmid: The sortase gene encodes an N-terminally truncatedStaphylococcus aureus sortase A (60-206) molecule. The expressionplasmid for the transient expression of soluble sortase in HEK293 cellscomprised besides the soluble sortase expression cassette an origin ofreplication from the vector pUC18, which allows replication of thisplasmid in E. coli, and a beta-lactamase gene which confers ampicillinresistance in E. coli. The transcription unit of the soluble sortasecomprises the following functional elements:

-   -   the immediate early enhancer and promoter from the human        cytomegalovirus (P-CMV) including intron A,    -   a human heavy chain immunoglobulin 5′-untranslated region        (5′UTR),    -   a murine immunoglobulin heavy chain signal sequence,    -   an N-terminally truncated S. aureus sortase A encoding nucleic        acid, and    -   the bovine growth hormone polyadenylation sequence (BGH pA).        Renaturation of Fab-PE24 Derived from E. coli Inclusion Bodies

Inclusion bodies of VH-PE24 and VL-C_(kappa) were solubilized separatelyin 8 M guanidinium hydrochloride, 100 mM Tris-HCl, 1 mM EDTA, pH 8.0+100mM dithiothreitol (DTT). After 12-16 hours at RT the pH of thesolubilisates was adjusted to 3.0, the centrifuged solutions weredialyzed against 8 M guanidinium hydrochloride, 10 mM EDTA, pH 3.0. Theprotein concentration was determined by Biuret reaction, the purity ofinclusion body preparations was estimated by SDS PAGE. Equimolar amountsof both chains were diluted in two steps into 0.5 M arginine, 2 mM EDTA,pH 10+1 mM GSH/1 mM GSSG, to a final concentration of 0.2-0.3 mg/ml.After 12-16 h at 4-10° C. the renaturated protein was diluted with H₂Oto <3 mS/cm and loaded onto a Q sepharose FF (GE healthcare)equilibrated in 20 mM Tris/HCl, pH 7.4. Elution was performed with agradient up to 400 mM NaCl in the same buffer. Fractions containing thecorrect product were identified by SDS-PAGE and analytical sizeexclusion chromatography (SEC). Pooled fractions were concentrated andloaded onto a HiLoad™ Superdex™ 200 (GE Healthcare) in 20 mM Tris, 150mM NaCl, pH 7.4 or alternatively in 20 mM histidine, 140 mM NaCl, pH6.0. Fractions were analyzed and pooled according to analytical SEC andstored at −80° C.

Based on SEQ ID NO:46 and 49 the immunoconjugate of Fab fragment ofM-05-74 with Pseudomonas exotoxin variant PE24LR8M (M-05-74-PE) can beexpressed recombinately, purified and renturated also as direct PE24LR8Mfusion.

Example 11

Cell Killing of Different Tumor Cell Lines by M-05-74-Fab-PseudomonasExotoxin Conjugate (M-05-74-PE)

HER3 overexpressing A549 cells were seeded into a white 96-well-plate(flat, transparent bottom, 1×10⁴ cells per well) and were grown in RPMI(10% FCS) overnight. On the next day, the media was exchanged by 50 μlstarving media (RPMI, 0.5% FCS). After at least 4 hours, 5 μlHeregulin-beta (PeproTech, Cat. #100-03) (HRG beta) was added to a finalconcentration of 500 ng/ml. 50 μl Fab-74-PE solution was added to finalconcentrations of 10, 3.3, 1.1, 0.37, 0.12, 0.04, 0.014, 0.005 and 0.002μg/ml. Plates were incubated for 72 h. After 24 h and 48 h, 5 μlHeregulin-beta was added again to a final concentration of 500 ng/ml.After 72 h the luminescence was measured in a Tecan Infinite F200 Readerusing the CellTiter-Glo Luminescent Cell Viability Assay by Promega(Cat. #G7571). The EC50 value for M-05-74-Fab-Pseudomonas exotoxinconjugate (M-05-74-PE) in the abscence of HRG beta was: 1,93 μg/ml andin the presence 0.13 μg/ml.

TABLE 10 EC50 of Cell killing of A549 cells by M-05-74-Fab-Pseudomonaspresence (+)/ absence (−) of ligand EC50 of (M- Heregulin-beta 05-74-PE)half max. (HRG) (μg/ml) inhibition +HRG beta 0.13 30.3 −HRG beta 1.9319.85

Example 12

Humanized Variants of the Antibodies According to the Invention

The binding specificity of the murine antibody is transferred onto ahuman acceptor framework to eliminate potential immunogenicity issuesarising from sequence stretches that the human body will recognize asforeign. This is done by engrafting the entire HVRs of the murine(donor) antibody onto a human (acceptor) antibody framework, and iscalled HVR (or CDR)-grafting or antibody humanization.

The murine variable region amino acid sequence is aligned to acollection of human germline antibody V-genes, and sorted according tosequence identity and homology. The acceptor sequence is selected basedon high overall sequence homology and optionally also the presence ofthe right canonical residues already in the acceptor sequence (see Poul,M-A. and Lefranc, M-P., in “Ingénierie des anticorps banquescombinatores” ed. by Lefranc, M-P. and Lefranc, G., Les Editions INSERM,1997).

The germline V-gene encodes only the region up to the beginning of HVR3for the heavy chain, and till the middle of HVR3 of the light chain.Therefore, the genes of the germline V-genes are not aligned over thewhole V-domain. The humanized construct comprises the human frameworks 1to 3, the murine HVRs, and the human framework 4 sequence derived fromthe human JK4, and the JH4 sequences for light and heavy chain,respectively.

Before selecting one particular acceptor sequence, the so-calledcanonical loop structures of the donor antibody can be determined (seeMorea, V., et al., Methods, Vol 20, Issue 3 (2000) 267-279). Thesecanonical loop structures are determined by the type of residues presentat the so-called canonical positions. These positions lie (partially)outside of the HVR regions, and should be kept functionally equivalentin the final construct in order to retain the HVR conformation of theparental (donor) antibody.

In WO 2004/006955 a method for humanizing antibodies is reported thatcomprises the steps of identifying the canonical HVR structure types ofthe HVRs in a non-human mature antibody; obtaining a library of peptidesequence for human antibody variable regions; determining the canonicalHVR structure types of the variable regions in the library; andselecting the human sequences in which the canonical HVR structure isthe same as the non-human antibody canonical HVR structure type atcorresponding locations within the non-human and human variable regions.

Summarizing, the potential acceptor sequence is selected based on highoverall homology and optionally in addition the presence of the rightcanonical residues already in the acceptor sequence.

In some cases simple HVR grafting only result in partial retention ofthe binding specificity of the non-human antibody. It has been foundthat at least some specific non-human framework residues are requiredfor reconstituting the binding specificity and have also to be graftedinto the human framework, i.e. so called “back mutations” have to bemade in addition to the introduction of the non-human HVRs (see e.g.Queen et al., Proc. Natl. Acad. Sci. USA 86 (1989) 10,029-10,033; Co etal., Nature 351 (1991) 501-502). These specific framework amino acidresidues participate in FR-HVR interactions and stabilized theconformation (loop) of the HVRs (see e.g. Kabat et al., J. Immunol. 147(1991) 1709).

In some cases also forward-mutations are introduced in order to adoptmore closely the human germline sequence.

The genes for those designed antibody sequences are generated byconventional PCR techniques. The heavy chain variable region is fused toeither the human IgG1 heavy chain constant region (if effector functionis required) or to a human IgG1/IgG4 heavy chain constant region variant(if no effector function is required; IgG1 L234A L235A P329G; IgG4 S228PL235E P329G). The light chain variable domain is fused to either thelight chain kappa or lambda constant domain for the construction of theexpression plasmids.

Accordingly the mouse anti-HER3/HER4 antibody M-05-74 was humanized togive the following humanized variants of M-05-74:

TABLE 11 VH and VL sequences of humanized variant antibodies of M-05-74humanized variant Humanized of light chain variable antibodies of M-humanized variant of domain VL//SEQ 05-74 VH/SEQ ID NO: ID NO: huMabM-05-74_1 <Her3> M-05-74_VH1 <Her3> M-05-74_VL1 SEQ ID NO: 33 SEQ ID NO:36 huMab M-05-74_2 <Her3> M-05-74_VH1 <Her3> M-05-74_VL2 SEQ ID NO: 33SEQ ID NO: 37 huMab M-05-74_3 <Her3> M-05-74_VH2 <Her3> M-05-74_VL1 SEQID NO: 34 SEQ ID NO: 36 huMab M-05-74_4 <Her3> M-05-74_VH2 <Her3>M-05-74_VL2 SEQ ID NO: 34 SEQ ID NO: 37 huMab M-05-74_5 <Her3>M-05-74_VH3 <Her3> M-05-74_VL1 SEQ ID NO: 35 SEQ ID NO: 36 huMabM-05-74_6 <Her3> M-05-74_VH3 <Her3> M-05-74_VL2 SEQ ID NO: 35 SEQ ID NO:37

TABLE 12 HVR sequences of humanized variant antibodies of M-05-74Humanized HVR-H1, HVR-H2, and HVR-L1, HVR-L2, and antibodies of M-HVR-H3 of humanized HVR-L3 of humanized 05-74 variant/SEQ ID NO:variant/SEQ ID NO: huMab humanized variant 1 humanized variant 1 HVR-M-05-74_1 HVR-H1_V1 (SEQ ID L1_V1 (SEQ ID NO: 40), NO: 38), humanizedvariant 1 HVR- humanized variant 1 L2_V1 (SEQ ID NO: 41), HVR-H2_V1 (SEQID HVR-L3 (SEQ ID NO: 30) NO: 39), HVR-H3 (SEQ ID NO: 27) huMabhumanized variant 1 humanized variant 1 HVR- M-05-74_2 HVR-H1_V1 (SEQ IDL1_V1 (SEQ ID NO: 40), NO: 38), humanized variant 1 HVR- humanizedvariant 1 L2_V1 (SEQ ID NO: 42), HVR-H2_V1 (SEQ ID HVR-L3 (SEQ ID NO:30) NO: 39), HVR-H3 (SEQ ID NO: 27) huMab HVR-H1 (SEQ ID NO: humanizedvariant 1 HVR- M-05-74_3 25), L1_V1 (SEQ ID NO: 40), humanized variant 1humanized variant 1 HVR- HVR-H2_V1 (SEQ ID L2_V1 (SEQ ID NO: 41), NO:39), HVR-L3 (SEQ ID NO: 30) HVR-H3 (SEQ ID NO: 27) huMab HVR-H1 (SEQ IDNO: humanized variant 1 HVR- M-05-74_4 25), L1_V1 (SEQ ID NO: 40),humanized variant 1 humanized variant 1 HVR- HVR-H2_V1 (SEQ ID L2_V1(SEQ ID NO: 42), NO: 39), HVR-L3 (SEQ ID NO: 30) HVR-H3 (SEQ ID NO: 27)huMab HVR-H1 (SEQ ID NO: humanized variant 1 HVR- M-05-74_5 25), L1_V1(SEQ ID NO: 40), humanized variant 1 humanized variant 1 HVR- HVR-H2_V1(SEQ ID L2_V1 (SEQ ID NO: 41), NO: 39), HVR-L3 (SEQ ID NO: 30) HVR-H3(SEQ ID NO: 27) huMab HVR-H1 (SEQ ID NO: humanized variant 1 HVR-M-05-74_6 25), L1_V1 (SEQ ID NO: 40), humanized variant 1 humanizedvariant 1 HVR- HVR-H2_V1 (SEQ ID L2_V1 (SEQ ID NO: 42), NO: 39), HVR-L3(SEQ ID NO: 30) HVR-H3 (SEQ ID NO: 27)

Antibodies were expressed in mammalian cell culture systems like HEK orCHO, and purified via protein A and size exclusion chromatography.Humanized antibodies were either expressed full-length antibodies orantibody fragments or are included in immunotoxin conjugates (seeExample 10). Humanized variants were evaluated with respect to theirbinding and biological properties as described above.

Binding to Binding to HER3-ECD HER3-ECD-HRG Antibody KD (bindingaffinity) KD (binding affinity) huMab M-05-74_5 3.2 nM 4.1 nM huMabM-05-74_6 8.3 nM 8.6 nM

Example 13

In Vivo Tumor Cell Growth Inhibition by M-05-74-Fab-Pseudomonas ExotoxinConjugate (M-05-74-PE)

The human A431-B34 non-small cell lung cancer cell line cell line, whichwas stably transfected with an expression vector encoding human HER3,was subcutaneously inoculated into the right flank of female SCID beigemice (1×10⁷ cells per animal).

On day 21 after tumor inoculation, the animals were randomized andallocated into the treatment group and one vehicle group, resulting in amedian tumor volume of ˜110 mm³ per group. On the same day, animals weretreated intravenously for 2 cycles, each cycle consisting of 3q7d (everyother day), with M-05-74-Fab-Pseudomonas exotoxin conjugate (M-05-74-PE)(1.0 mg/kg). Controls received vehicle (Tris buffer). The two cycleswere separated by a one week off-treatment.

Primary tumor volume (TV) was calculated according to the NCI protocol(TV=(length×width²)/2), where “length” and “width” are long and shortdiameters of tumor mass in mm (Corbett et al., 1997). Calculation wasexecuted from staging (day 21 after tumor inoculation) until day 42after tumor inoculation, and values were documented as medians andinter-quartile ranges (IQR) defined as differences of the third andfirst quartile.

For calculation of percentage tumor growth inhibition (TGI) during thetreatment period, every treated group was compared with its respectivevehicle control. TV_(day z) represents the tumor volume of an individualanimal at a defined study day (day z) and TV_(day x) represents thetumor volume of an individual animal at the staging day (day x).

The following formula was applied:

${{TGI}\lbrack\%\rbrack} = {100 - {\frac{{median}\left( {{{TV}({treated})}_{{day}\mspace{11mu} z} - {{TV}({treated})}_{{day}\mspace{11mu} x}} \right)}{{median}\left( {{{TV}\left( {{resp}.\mspace{11mu}{control}} \right)}_{{day}\mspace{11mu} z} - {{TV}\left( {{resp}.\mspace{11mu}{control}} \right)}_{{day}\mspace{11mu} x}} \right)} \times 100}}$

Calculations of treatment to control ratio (TCR) with confidenceinterval (CI) were applied using non-parametric methods. Results ofmedian tumor volumes with inter-quartile ranges are shown in FIG. 19.Tumor growth inhibition was 66% of M-05-74-Fab-Pseudomonas exotoxinconjugate (M-05-74-PE) with a TCR of 0.509 (CI:0.33-0.734).

Example 14

Binding of the Antibody M-05-74 (1) to TtSlyDcys-Her3 (SEQ ID NO: 18) inComparison with Anti-HER3 Antibody MOR09823 (2) Described inWO2012/22814.

A Biacore T200 instrument (GE Healthcare) was mounted with CMS seriessensor and was normalized in HBS-ET+ buffer (10 mM HEPES pH 7.4, 150 mMNaCl, 3 mM EDTA, 0.05% w/v Tween 20) according to the manufacturer'sinstructions. The sample buffer was the system buffer supplemented with1 mg/ml CMD (Carboxymethyldextran). The system operated at 37° C. Adouble antibody capture system was established on the sensor surface.6500 RU mAb<M-IgG>R was immobilized according to the manufacturer'sinstructions using EDC/NHS chemistry on all flow cells. The sensor wasdeactivated using 1M ethanolamine. Flow cell 1 served as a reference andwas captured for 1 min at 10 μl/min with anti-TSH IgG1 antibody. On flowcell 2 M-5-74 was captured for 1 min at 10μl/min. On flow cell 3 amurine anti-human FC pan antibody was captured 1 min at 10 μl/minfollowed by the injection of the anti-HER3 antibody M-05-74 (1) or ofanti-HER3 antibody MOR09823 antibody for 1 min at 10 μl/min. The flowrate was set to 60 μ/min. The analyte in solution TtSlyDcys-HER3 (SEQ IDNO: 18) was injected at concentrations of 0 nM and 150 nM for 5 min andthe dissociation was monitored for 600 sec. The sensor was fullyregenerated by one injection at 10 μl/min for 3 min with 10 mM glycinepH 1.7 buffer.

FIG. 20 depicts a sensorgram overlay plot showing binding signals at 150nM of, TtSlyDcys-Her3 and buffer. The overlay plot above shows theantibody M-5-74 binding at 150 nM TtSlyDcys-Her3 (1). MOR09823 antibodydoes not bind TtSlyDcas-Her3 (2). (3) shows the background bindingsignal of the TtSlyDcas-HER3 versus the mAb<M-IgG>R capture surface. Theanti-HER3 antibody MOR09823 (2) described in WO2012/22814 does not showany interaction at 150 nM TtSlyDcys-Her3. The positive control antibodyM-05-74 (1) shows significant binding versus TtSlyDcas-Her3. Nointeraction could be determined with both antibodies when injecting 150nM TtSlyDcys (no HER-3 insertion) (data not shown).

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

We claim:
 1. An isolated nucleic acid encoding an antibody that binds tohuman HER3 and binds to human HER4, wherein the antibody comprises: (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:25, (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:26, (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:27, (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:28, (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:29, and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:30.
 2. An isolatednucleic acid encoding an antibody that binds to human HER3 and binds tohuman HER4, wherein the antibody is selected from the group consistingof i) an antibody comprising: (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO:38, (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO:39, (c) HVR-H3 comprising the amino acid sequence of SEQ IDNO:27, (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:40,(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:41, and (f)HVR-L3 comprising the amino acid sequence of SEQ ID NO:30; ii) anantibody comprising: (a) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:38, (b) HVR-H2 comprising the amino acid sequence of SEQ IDNO:39, (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:27,(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:40, (e)HVR-L2 comprising the amino acid sequence of SEQ ID NO:42, and (f)HVR-L3 comprising the amino acid sequence of SEQ ID NO:30; iii) anantibody comprising: (a) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:25, (b) HVR-H2 comprising the amino acid sequence of SEQ IDNO:39, (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:27,(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:40, (e)HVR-L2 comprising the amino acid sequence of SEQ ID NO:41, and (f)HVR-L3 comprising the amino acid sequence of SEQ ID NO:30; and iv) anantibody comprising: (a) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:25, (b) HVR-H2 comprising the amino acid sequence of SEQ IDNO:39, (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:27,(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:40, (e)HVR-L2 comprising the amino acid sequence of SEQ ID NO:42, and (f)HVR-L3 comprising the amino acid sequence of SEQ ID NO:30.
 3. Anisolated nucleic acid encoding an antibody that binds to human HER3 andbinds to human HER4, wherein the antibody is selected from the groupconsisting of a) an antibody comprising a VH sequence of SEQ ID NO:33and a VL sequence of SEQ ID NO:36; b) an antibody comprising a VHsequence of SEQ ID NO:33 and a VL sequence of SEQ ID NO:37; c) anantibody comprising a VH sequence of SEQ ID NO:34 and a VL sequence ofSEQ ID NO:36; d) an antibody comprising a VH sequence of SEQ ID NO:34and a VL sequence of SEQ ID NO:37; e) an antibody comprising a VHsequence of SEQ ID NO:35 and a VL sequence of SEQ ID NO:36; and f) anantibody comprising a VH sequence of SEQ ID NO:35 and a VL sequence ofSEQ ID NO:37.
 4. The isolated nucleic acid of claim 2, wherein nucleicacid encodes an antibody comprising (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO:38, (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO:39, (c) HVR-H3 comprising the amino acid sequence of SEQ IDNO:27, (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:40,(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:41, and (f)HVR-L3 comprising the amino acid sequence of SEQ ID NO:30.
 5. Theisolated nucleic acid of claim 2, wherein nucleic acid encodes anantibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQID NO:38, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:39,(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:27, (d)HVR-L1 comprising the amino acid sequence of SEQ ID NO:40, (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:42, and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:30.
 6. The isolatednucleic acid of claim 2, wherein nucleic acid encodes an antibodycomprising (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO:25, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:39,(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:27, (d)HVR-L1 comprising the amino acid sequence of SEQ ID NO:40, (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:41, and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:30.
 7. The isolatednucleic acid of claim 2, wherein nucleic acid encodes an antibodycomprising (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO:25, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:39,(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:27, (d)HVR-L1 comprising the amino acid sequence of SEQ ID NO:40, (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:42, and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:30.
 8. The isolatednucleic acid of claim 3, wherein nucleic acid encodes an antibodycomprising a VH sequence of SEQ ID NO:33 and a VL sequence of SEQ IDNO:36.
 9. The isolated nucleic acid of claim 3, wherein nucleic acidencodes an antibody comprising a VH sequence of SEQ ID NO:33 and a VLsequence of SEQ ID NO:37.
 10. The isolated nucleic acid of claim 3,wherein nucleic acid encodes an antibody comprising a VH sequence of SEQID NO:34 and a VL sequence of SEQ ID NO:36.
 11. The isolated nucleicacid of claim 3, wherein nucleic acid encodes an antibody comprising aVH sequence of SEQ ID NO:34 and a VL sequence of SEQ ID NO:37.
 12. Theisolated nucleic acid of claim 3, wherein nucleic acid encodes anantibody comprising a VH sequence of SEQ ID NO:35 and a VL sequence ofSEQ ID NO:36.
 13. The isolated nucleic acid of claim 3, wherein nucleicacid encodes an antibody comprising a VH sequence of SEQ ID NO:35 and aVL sequence of SEQ ID NO:37.
 14. A host cell comprising the nucleic acidof any one of claims 1-13.
 15. A method of producing an antibodycomprising culturing the host cell of claim 11 so that the antibody isproduced, and recovering said antibody from said cell culture or thecell culture supernatant.
 16. The method of claim 15 further comprisingrecovering said antibody from said cell culture or the cell culturesupernatant.